Method and instrument for detecting biomolecular interactions

ABSTRACT

Method and apparatus for detecting biomolecular interactions. The use of labels is not required and the methods may be performed in a high-throughput manner. An instrument system for detecting a biochemical interaction on a biosensor. The system includes an array of detection locations comprises a light source for generating collimated white light. A beam splitter directs the collimated white light towards a surface of a sensor corresponding to the detector locations. A detection system includes an imaging spectrometer receiving the reflected light and generating an image of the reflected light.

A. PRIORITY

[0001] This application claims the benefit of U.S. provisionalapplication No. 60/244,312 filed Oct. 30, 2000; U.S. provisionalapplication No. 60/283,314 filed Apr. 12, 2001; U.S. provisionalapplication No. 60/303,028 filed Jul. 3, 2001; and is acontinuation-in-part of U.S. patent application Ser. No. 09/930,352filed Aug. 15, 2001, U.S. patent application Ser. No. 10/059,060 filedJan. 28, 2002, and U.S. patent application Ser. No. 10/058,626 filedJan. 28, 2002, all of which are herein entirely incorporated byreference and to which the reader is directed for further information.

B. TECHNICAL AREA OF THE INVENTION

[0002] The invention generally relates to methods, instrumentation anddevices for detecting biomolecular interactions.

C. BACKGROUND OF THE INVENTION

[0003] With the completion of the sequencing of the human genome, one ofthe next grand challenges of molecular biology will be to understand howthe many protein targets encoded by DNA interact with other proteins,small molecule pharmaceutical candidates, and a large host of enzymesand inhibitors. See e.g., Pandey & Mann, “Proteomics to study genes andgenomes,” Nature, 405, p. 837-846, 2000; Leigh Anderson et al.,“Proteomics: applications in basic and applied biology,” Current Opinionin Biotechnology, 11, p. 408-412, 2000; Patterson, “Proteomics: theindustrialization of protein chemistry,” Current Opinion inBiotechnology, 11, p. 413-418, 2000; MacBeath & Schreiber, “PrintingProteins as Microarrays for High-Throughput Function Determination,”Science, 289, p. 1760-1763, 2000; De Wildt et al., “Antibody arrays forhigh-throughput screening of antibody-antigen interactions,” NatureBiotechnology, 18, p. 989-994, 2000. To this end, tools that have theability to simultaneously quantify many different biomolecularinteractions with high sensitivity will find application inpharmaceutical discovery, proteomics, and diagnostics. Further, forthese tools to find widespread use, they must be simple to use,inexpensive to own and operate, and applicable to a wide range ofanalytes that can include, for example, polynucleotides, peptides, smallproteins, antibodies, and even entire cells.

[0004] Biosensors have been developed to detect a variety ofbiomolecular complexes including oligonucleotides, antibody-antigeninteractions, hormone-receptor interactions, and enzyme-substrateinteractions. In general, biosensors consist of two components: a highlyspecific recognition element and a transducer that converts themolecular recognition event into a quantifiable signal. Signaltransduction has been accomplished by many methods, includingfluorescence, interferometry (Jenison et al., “Interference-baseddetection of nucleic acid targets on optically coated silicon,” NatureBiotechnology, 19, p. 62-65; Lin et al., “A porous silicon-based opticalinterferometric biosensor,” Science, 278, p. 840-843, 1997), andgravimetry (A. Cunningham, Bioanalytical Sensors, John Wiley & Sons(1998)).

[0005] Of the optically-based transduction methods, direct methods thatdo not require labeling of analytes with fluorescent compounds are ofinterest due to the relative assay simplicity and ability to study theinteraction of small molecules and proteins that are not readilylabeled. Direct optical methods include surface plasmon resonance (SPR)(Jordan & Corn, “Surface Plasmon Resonance Imaging Measurements ofElectrostatic Biopolymer Adsorption onto Chemically Modified GoldSurfaces,” Anal. Chem., 69:1449-1456 (1997), (grating couplers (Morhardet al., “Immobilization of antibodies in micropatterns for celldetection by optical diffraction,” Sensors and Actuators B, 70, p.232-242, 2000), ellipsometry (Jin et al., “A biosensor concept based onimaging ellipsometry for visualization of biomolecular interactions,”Analytical Biochemistry, 232, p. 69-72, 1995), evanascent wave devices(Huber et al., “Direct optical immunosensing (sensitivity andselectivity),” Sensors and Actuators B, 6, p. 122-126, 1992), andreflectometry (Brecht & Gauglitz, “Optical probes and transducers,”Biosensors and Bioelectronics, 10, p. 923-936, 1995). Theoreticallypredicted detection limits of these detection methods have beendetermined and experimentally confirmed to be feasible down todiagnostically relevant concentration ranges. However, to date, thesemethods have yet to yield commercially available high-throughputinstruments that can perform high sensitivity assays without any type oflabel in a format that is readily compatible with the microtiterplate-based or microarray-based infrastructure that is most often usedfor high-throughput biomolecular interaction analysis. Therefore, thereis a need in the art for compositions and methods that can achieve thesegoals.

D. SUMMARY OF THE INVENTION

[0006] It is an object of the invention to provide methods,instrumentation and devices for detecting binding of one or morespecific binding substances to their respective binding partners. Thisand other objects of the invention are provided by one or more of theembodiments described below.

[0007] In one arrangement, an instrument system for detecting abiochemical interaction on a biosensor comprising an array of detectionlocations comprises a light source for generating collimated whitelight. A beam splitter directs the collimated white light towards asurface of a sensor corresponding to the detector locations. A detectionsystem includes an imaging spectrometer receiving the reflected lightand generating an image of the reflected light.

[0008] In an alternative arrangement, an instrument for calculating apeak wavelength comprises an incubator assembly for incubating abiosensor. An optical assembly illuminates the biosensor with light andcollects reflected radiation from the biosensor. A spectrometer receivesthe said reflected radiation and software derives a peak wavelength fromthe reflected and detected wavelength.

[0009] Unlike surface plasmon resonance, resonant mirrors, and waveguidebiosensors, the described compositions and methods enable many thousandsof individual binding reactions to take place simultaneously upon thebiosensor surface. This technology is useful in applications where largenumbers of biomolecular interactions are measured in parallel,particularly when molecular labels alter or inhibit the functionality ofthe molecules under study. High-throughput screening of pharmaceuticalcompound libraries with protein targets, and microarray screening ofprotein-protein interactions for proteomics are examples of applicationsthat require the sensitivity and throughput afforded by this approach. Abiosensor of the invention can be manufactured, for example, in largeareas using a plastic embossing process, and thus can be inexpensivelyincorporated into common disposable laboratory assay platforms such asmicrotiter plates and microarray slides.

[0010] These as well as other features and advantages of the presentinvention will become apparent to those of ordinary skill in the art byreading the following detailed description, with appropriate referenceto the accompanying drawings.

E. BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1A illustrates a schematic diagram of an embodiment of anoptical grating structure.

[0012]FIG. 1B illustrates a perspective view of the optical gratingstructure illustrated in FIG. 1A.

[0013]FIG. 2 illustrates a schematic drawing of a linear gratingstructure.

[0014]FIG. 3A illustrates a 2-D biosensor grating comprising a grid ofsquares/rectangles.

[0015]FIG. 3B illustrates a 2-D biosensor grating comprising a grid ofcircular holes.

[0016]FIG. 4 illustrates an embodiment of a biosensor utilizing asinusoidally varying grating profile.

[0017]FIG. 5(a) illustrates an alternative embodiment of a biosensorutilizing an embossed substrate.

[0018]FIG. 5(b) illustrates an alternative embodiment of a biosensorutilizing a plurality of concentric rings.

[0019]FIG. 5(c) illustrates an alternative embodiment of a biosensorhaving an array of closely packed hexagons.

[0020]FIG. 6A illustrates a transparent resonant reflectionsuperstructure arrangement.

[0021]FIG. 6B illustrates an alternative superstructure arrangement.

[0022]FIG. 6C illustrates a reflective surface of an alternativeembodiment of a biosensor.

[0023]FIG. 7 illustrates an alternative embodiment of a biosensorgrating structure.

[0024]FIG. 8A illustrates a biosensor embodiment incorporated into amicrotiter plate.

[0025]FIG. 8B illustrates a microarray slide that may be utilized withthe microtiter plate embodiment illustrated in FIG. 8A.

[0026]FIG. 9 illustrates an embodiment of a biosensor platform toperform assays.

[0027]FIG. 10 illustrates an embodiment of an array of biosensorelectrodes.

[0028]FIG. 11 illustrates a SEM photograph showing a plurality ofseparate grating regions of an array of biosensor electrodes.

[0029]FIG. 12(a) illustrates an embodiment of a biosensor upper surfaceimmersed in a liquid sample.

[0030]FIG. 12(b) illustrates an attraction of electronegative moleculesto a biosensor surface when a positive voltage is applied to thebiosensor illustrated in FIG. 12(a).

[0031]FIG. 12(c) illustrates an application of a repelling force such asa reversed electrical charge to electronegative molecules when anegative electrode voltage is applied to the biosensor illustrated inFIG. 12(a).

[0032]FIG. 13 illustrates an embodiment of a detecting system.

[0033]FIG. 14 illustrates resonance wavelength of a biosensor as afunction of incident angle of detection beam.

[0034]FIG. 15 illustrates an alternative embodiment of a detectionsystem that includes a beam splitter.

[0035]FIG. 16 illustrates an embodiment of a biosensor incorporating anITO grating.

[0036]FIG. 17 illustrates an optical fiber probe measuring apparatus.

[0037]FIG. 18(a) illustrates an optical fiber probe that may be utilizedwith the optical fiber probe measuring apparatus illustrated in FIG. 17.

[0038]FIG. 18(b) illustrates a general arrangement of a CCD chip and aspectrometer.

[0039]FIG. 18(c) illustrates a readout of a grating and the CCD chipillustrated in FIG. 18(b).

[0040]FIG. 19 illustrates an alternative embodiment of a measuringapparatus.

[0041]FIG. 20 illustrates yet another alternative embodiment of ameasuring apparatus.

[0042]FIG. 21 illustrates an alternative embodiment of an instrument fordetection of biomolecular interactions in accordance with one possibleembodiment.

[0043]FIG. 22 illustrates a perspective view of the measuring apparatusillustrated in FIG. 21.

[0044]FIG. 23 illustrates a perspective view of the transition stageassembly illustrated in FIG. 22.

[0045]FIG. 24 illustrates a perspective view of the transition stageassembly of FIG. 23 with an incubator assembly top portion removed.

[0046]FIG. 25 illustrates a visualization of a spotted micro-arrayimage.

F. DETAILED DESCRIPTION OF THE INVENTION

[0047] 1. Overview of Method and System

[0048] The present invention generally relates to a method and systemfor detecting biomolecular interactions. Preferably, these biomolecularinteractions occur on a subwavelength structured surface biosensor, asdescribed below.

[0049] One aspect of the present invention relates to a method andapparatus for detecting biochemical interactions occurring on a surfaceof a biosensor. In one embodiment, the biosensor is a colorimetricresonant optical biosensor embedded into a surface of a microarrayslide, microtiter plate or other device.

[0050] The colorimetric resonant optical biosensor allows biochemicalinteractions to be measured on the sensor's surface without the use offluorescent tags or colorimetric labels. The sensor surface contains anoptical structure that, when illuminated with collimated white light, isdesigned to reflect only a narrow band of wavelengths. The narrowwavelength is described as a wavelength “peak.” The “peak wavelengthvalue” (PWV) changes when biological material is deposited or removedfrom the sensor surface.

[0051] A disclosed measurement instrument performs a number offunctions, in addition to detection of a peak wavelength value. Forexample, the instrument can incubate a microtiter plate incorporatingthe biosensor plate at a user determined temperature. The instrument mayalso provide a mechanism for mixing samples within the microtiter platewells while the microtiter plate resides within the instrument.

[0052] In one possible embodiment, the instrument illuminates thebiosensor surface by directing a collimated white light on to the sensorstructure. The illuminated light may take the form of a spot ofcollimated light. Alternatively, the light is generated in the form of afan beam.

[0053] The instrument collects light reflected from the illuminatedbiosensor surface. The instrument may gather this reflected light frommultiple locations on the biosensor surface simultaneously. Theinstrument can include a plurality of illumination probes that directthe light to a discrete number of positions across the biosensorsurface. The instrument measures the Peak Wavelength Values (PWVs) ofseparate locations within the biosensor-embedded microtiter plate usinga spectrometer. In one embodiment, the spectrometer is a single-pointspectrometer. Alternatively, an imaging spectrometer is used.

[0054] The spectrometer produces a PWV image map of the sensor surface.In one embodiment, the measuring instrument spatially resolves PWVimages with less than 200 micron resolution.

[0055] 2. Subwavelength Structured Surface (SWS) Biosensor

[0056] In one embodiment of the present invention, a subwavelengthstructured surface (SWS) may be used to create a sharp optical resonantreflection at a particular wavelength that can be used to track withhigh sensitivity the interaction of biological materials, such asspecific binding substances or binding partners or both. A colormetricresonant diffractive grating surface acts as a surface binding platformfor specific binding substances.

[0057] Subwavelength structured surfaces are an unconventional type ofdiffractive optic that can mimic the effect of thin-film coatings. (Peng& Morris, “Resonant scattering from two-dimensional gratings,” J. Opt.Soc. Am. A, Vol. 13, No. 5, p. 993, May; Magnusson, & Wang, “Newprinciple for optical filters,” Appl. Phys. Lett., 61, No. 9, p. 1022,August, 1992; Peng & Morris, “Experimental demonstration of resonantanomalies in diffraction from two-dimensional gratings,” Optics Letters,Vol. 21, No. 8, p. 549, April, 1996). A SWS structure contains asurface-relief, two-dimensional grating in which the grating period issmall compared to the wavelength of incident light so that nodiffractive orders other than the reflected and transmitted zerothorders are allowed to propagate. A SWS surface narrowband filter cancomprise a two-dimensional grating sandwiched between a substrate layerand a cover layer that fills the grating grooves. Optionally, a coverlayer is not used. When the effective index of refraction of the gratingregion is greater than the substrate or the cover layer, a waveguide iscreated.

[0058] When a filter is designed according to one aspect of the presentinvention, incident light passes into the waveguide region. Atwo-dimensional grating structure selectively couples light at a narrowband of wavelengths into the waveguide. The light propagates only ashort distance (on the order of 10-100 micrometers), undergoesscattering, and couples with the forward- and backward-propagatingzeroth-order light. This sensitive coupling condition can produce aresonant grating effect on the reflected radiation spectrum, resultingin a narrow band of reflected or transmitted wavelengths. The depth andperiod of the two-dimensional grating are less than the wavelength ofthe resonant grating effect.

[0059] The reflected or transmitted color of this structure can bemodulated by the addition of molecules such as specific bindingsubstances or binding partners or both to the upper surface of the coverlayer or the two-dimensional grating surface. The added moleculesincrease the optical path length of incident radiation through thestructure, and thus modify the wavelength at which maximum reflectanceor transmittance will occur.

[0060] In one embodiment, a biosensor, when illuminated with whitelight, is designed to reflect only a single wavelength. When specificbinding substances are attached to the surface of the biosensor, thereflected wavelength (color) is shifted due to the change of the opticalpath of light that is coupled into the grating. By linking specificbinding substances to a biosensor surface, complementary binding partnermolecules can be detected without the use of any kind of fluorescentprobe or particle label. The detection technique is capable of resolvingchanges of, for example, ˜0.1 nm thickness of protein binding, and canbe performed with the biosensor surface either immersed in fluid ordried. A more detailed description of these binding partners is providedin related and commonly assigned patent application Ser. No. 09/930,352,herein entirely incorporated by reference and to which the reader isdirected for further information.

[0061] In one embodiment of the present invention, a detection systemconsists of, for example, a light source that illuminates a small spotof a biosensor at normal incidence through, for example, a fiber opticprobe. A spectrometer collects the reflected light through, for example,a second fiber optic probe also at normal incidence. Because no physicalcontact occurs between the excitation/detection system and the biosensorsurface, no special coupling prisms are required. The biosensor can,therefore, be adapted to a commonly used assay platform including, forexample, microtiter plates and microarray slides. A spectrometer readingcan be performed in several milliseconds, thus it is possible toefficiently measure a large number of molecular interactions takingplace in parallel upon a biosensor surface, and to monitor reactionkinetics in real time.

[0062] This technology is useful in applications where large numbers ofbiomolecular interactions are measured in parallel, particularly whenmolecular labels would alter or inhibit the functionality of themolecules under study. High-throughput screening of pharmaceuticalcompound libraries with protein targets, and microarray screening ofprotein-protein interactions for proteomics are examples of applicationsthat require the sensitivity and throughput afforded by the compositionsand methods of the invention.

[0063] A schematic diagram of an example of a SWS structure 10 is shownin FIG. 1. In FIG. 1, n_(substrate) represents a refractive index of asubstrate material 12. n₁ represents the refractive index of an optionalcover layer 14. n₂ represents the refractive index of a two-dimensionalgrating 16. N_(b10) represents the refractive index of one or morespecific binding substances 20. t₁ represents the thickness of the coverlayer 14 above the two-dimensional grating structure 16. t₂ representsthe thickness of the grating. t_(b10) represents the thickness of thelayer of one or more specific binding substances 20. In one embodiment,n2>n1 (see FIG. 1). Layer thicknesses (i.e. cover layer 14, one or morespecific binding substances 20, or a two-dimensional grating 16) areselected to achieve resonant wavelength sensitivity to additionalmolecules on a top surface 15. A grating period is selected to achieveresonance at a desired wavelength.

[0064] One embodiment provides a SWS biosensor, such as the SWSbiosensor 10 illustrated in FIG. 1. The SWS biosensor 10 comprises atwo-dimensional grating 16 and a substrate layer 12 that supports thetwo-dimensional grating 16. One or more specific binding substances 20may be immobilized on a surface 15 of the two-dimensional grating 16opposite of the substrate layer 12. Incident light 11 is polarizedperpendicular to the grating structure and results in the reflectedlight 13. FIG. 1B illustrates a perspective view of the optical gratingstructure illustrated in FIG. 1A. Note that a SWS biosensor worksequally well whether illumination and reflection occur from the top ofthe sensor surface or from the bottom, although FIG. 1 illustrates onlythe bottom illumination and reflection case.

[0065] The two-dimensional grating 16 can comprise a material,including, for example, zinc sulfide, titanium dioxide, tantalum oxide,and silicon nitride. A cross-sectional profile of a two-dimensionalgrating can comprise any periodically repeating function, for example, a“square-wave.” The two-dimensional grating can comprise a repeatingpattern of shapes selected from the group consisting of lines, squares,circles, ellipses, triangles, trapezoids, sinusoidal waves, ovals,rectangles, and hexagons. A sinusoidal cross-sectional profile ispreferable for manufacturing applications that require embossing of agrating shape into a soft material such as plastic. In one embodiment ofthe biosensor, the depth of the grating is about 0.01 micron to about 1micron and the period of the grating is about 0.01 micron to about 1micron.

[0066] Linear gratings have resonant characteristics where theilluminating light polarization is oriented perpendicular to the gratingperiod. However, a hexagonal grid of holes has increased polarizationsymmetry over that of a rectangular grid of holes. Therefore, acolorimetric resonant reflection biosensor can comprise, for example, ahexagonal array of holes or alternatively, a grid of parallel lines.FIG. 3B illustrates a 2-D biosensor grating 34 including a grid ofcircular holes 36. FIG. 3A illustrates a 2-D biosensor grating 30including a grid of squares/rectangles 32.

[0067] A linear grating has the same pitch (i.e. distance betweenregions of high and low refractive index), period, layer thicknesses,and material properties as the hexagonal array grating. However, lightmust be polarized perpendicular to the grating lines in order to beresonantly coupled into the optical structure. Therefore, a polarizingfilter oriented with its polarization axis perpendicular to the lineargrating is inserted between the illumination source and the biosensorsurface. Because only a small portion of the illuminating light sourceis correctly polarized, a longer integration time is required to collectan equivalent amount of resonantly reflected light compared to ahexagonal grating.

[0068] While a linear grating can require either a higher intensityillumination source or a longer measurement integration time compared toa hexagonal grating, the fabrication requirements for the linearstructure are generally less complex. A hexagonal grating pattern isproduced by holographic exposure of photoresist to three mutuallyinterfering laser beams. The three beams are aligned in order to producea grating pattern that is essentially symmetrical in three directions. Alinear grating pattern requires alignment of only two laser beams toproduce a holographic exposure in photoresist, and therefore has areduced alignment requirement. A linear grating pattern can also beproduced by, for example, direct writing of photoresist with an electronbeam. Also, several commercially available sources exist for producinglinear grating “master” templates for embossing a grating structure intoplastic.

[0069] A schematic diagram of a linear grating structure 24 is shown inFIG. 2. The grating structure 24 includes a grating fill layer 25, agrating structural layer 26, and a substrate 27. Incident light 28 ispolarized perpendicular to the grating structure and results in thereflected light 29.

[0070] A rectangular grid pattern can be produced in photoresist usingan electron beam direct-write exposure system. A single wafer can beilluminated as a linear grating with two sequential exposures with thepart rotated 90-degrees between exposures.

[0071]FIG. 5(a) illustrates a two-dimensional grating 50.Two-dimensional grating 50 comprises a “stepped” profile 52. In such astepped profile 52, the profile has high refractive index regions of asingle, fixed height embedded within a lower refractive index coverlayer. The alternating regions of high and low refractive index providean optical waveguide parallel to a top surface of the biosensor.

[0072] For manufacture, a stepped structure, such as the structureillustrated in FIG. 5(a), is etched or embossed into a substratematerial 54 such as glass or plastic. A uniform thin film of higherrefractive index material 51, such as silicon nitride (SiN) or zincsulfide (ZnS) is deposited on this structure. The deposited layerfollows the contour of the embossed or etched structure in the substrate54. Consequently, the deposited material 51 has a surface relief profilethat is essentially identical to the original embossed or etched profileof embossed plastic substrate 54.

[0073] The structure 50 can be completed with an application of anoptional cover layer 56. Preferably, cover layer 56 includes a materialhaving a lower refractive index than the higher refractive indexmaterial and has a substantially flat upper surface 57. The coveringmaterial 56 can be, for example, glass, epoxy, or plastic.

[0074] A stepped structure, such as the structure illustrated in FIG.5(a), allows for reduced cost biosensor manufacturing, because thebiosensor can be mass produced. For example, a “master” grating can beproduced in glass, plastic, or metal using a three-beam laserholographic patterning process. See e.g., Cowan, The recording and largescale production of crossed holographic grating arrays using multiplebeam interferometry, Proc. Soc. Photo-optical Instum. Eng. 503:120(1984). A master grating can be repeatedly used to emboss a plasticsubstrate. The embossed substrate 50 is subsequently coated with a highrefractive index material and optionally, a cover layer 56.

[0075] While a stepped structure poses essentially few manufacturingcomplications, it is also possible to make a resonant biosensor in whicha high refractive index material is not stepped. Rather, a biosensorcould include a high refractive index material that varies with lateralposition. For example, FIG. 4 illustrates an alternative embodiment of abiosensor 40. In this embodiment, the biosensor 40 includes atwo-dimensional grating 42 having a high refractive index materialvarying with lateral position.

[0076] The biosensor 40 includes a substrate layer 41 that supports thetwo-dimensional grating 42. One or more specific binding substances 46may be immobilized on a surface 43 of the two-dimensional gratingopposite of the substrate layer 41. Sensor 40 includes a profile inwhich a high refractive index material of the two-dimensional grating42, n₂, sinusoidally varies in height. This varying height of thetwo-dimensional grating 42 is represented by t₂.

[0077] To produce a resonant reflection at a particular wavelength, theperiod of the sinusoidally varying grating 42 is essentially identicalto the period of an equivalent stepped structure. The resonant operationof the sinusoidally varying structure 40 and its functionality as abiosensor has been verified using GSOLVER (Grating Solver DevelopmentCompany, Allen, Tex., USA) computer models.

[0078] Techniques for making two-dimensional gratings are disclosed inWang, J. Opt. Soc. Am No. 8, August 1990, pp. 1529-44. Biosensors asherein described can be made in, for example, a semiconductormicrofabrication facility. Biosensors can also be made on a plasticsubstrate using continuous embossing and optical coating processes. Forthis type of manufacturing process, a “master” structure is built in arigid material such as glass or silicon. These “master” structures areused to generate “mother” structures in an epoxy or plastic using one ofseveral types of replication procedures. The “mother” structure, inturn, is coated with a thin film of conductive material, and used as amold to electroplate a thick film of nickel. The nickel “daughter” isreleased from the plastic “mother” structure. Finally, the nickel“daughter” is bonded to a cylindrical drum, which is used tocontinuously emboss the surface relief structure into a plastic film.

[0079] A device structure that uses an embossed plastic substrate isshown in FIG. 5(a). Following embossing, the plastic substrate 54 isovercoated with a thin film of high refractive index material 51. Thesubstrate 54 may be optionally coated with a planarizing, cover layerpolymer, and cut to appropriate size.

[0080] In one biosensor embodiment, a substrate for a SWS biosensorcomprises glass, plastic or epoxy. Alternatively, a substrate and atwo-dimensional grating comprise a single biosensor unit. That is, a twodimensional grating and substrate are formed from the same material,such as, for example, glass, plastic, or epoxy. The surface of a singleunit comprising the two-dimensional grating is coated with a materialhaving a high refractive index, for example, zinc sulfide, titaniumdioxide, tantalum oxide, and silicon nitride. One or more specificbinding substances can be immobilized on the surface of the materialhaving a high refractive index or on an optional cover layer.

[0081] An alternative biosensor embodiment can further comprise a coverlayer on the surface of a two-dimensional grating opposite of asubstrate layer. Where a cover layer is present, the one or morespecific binding substances are immobilized on the surface of the coverlayer opposite of the two-dimensional grating. Preferably, a cover layercomprises a material that has a lower refractive index than a materialthat comprises the two-dimensional grating. A cover layer can becomprised of, for example, glass (including spin-on glass (SOG)), epoxy,or plastic.

[0082] Various polymers that meet the refractive index requirement of abiosensor can be used for a cover layer. For example, SOG can be useddue to its favorable refractive index, ease of handling, and readinessof being activated with specific binding substances using a number ofknown glass surface activation techniques. When the flatness of thebiosensor surface is not an issue for a particular application, agrating structure of SiN/glass can directly be used as the sensingsurface, the activation of which can be done using the same means as ona glass surface.

[0083] Resonant reflection can also be obtained without a planarizingcover layer over a two-dimensional grating. For example, a biosensor cancontain only a substrate coated with a structured thin film layer ofhigh refractive index material. Without the use of a planarizing coverlayer, the surrounding medium (such as air or water) fills the grating.Therefore, specific binding substances are immobilized to the biosensoron all surfaces a two-dimensional grating exposed to the specificbinding substances, rather than only on an upper surface.

[0084] According to an alternative embodiment, a biosensor isilluminated with white light that contains light of every polarizationangle. The orientation of the polarization angle with respect torepeating features in a biosensor grating determines a resonancewavelength. For example, a “linear grating” biosensor structureconsisting of a set of repeating lines and spaces will have two opticalpolarizations that can generate separate resonant reflections. Lightthat is polarized perpendicularly to the lines is referred to as“s-polarized,” whereas light that is polarized parallel to the lines isreferred to as “p-polarized.” Both the s and the p components ofincident light exist simultaneously in an unfiltered illumination beam,and each component generates a separate resonant signal. A biosensorstructure can generally be designed to optimize the properties of onlyone polarization (the s-polarization), and the non-optimizedpolarization can be removed by a polarizing filter.

[0085] In order to remove the polarization dependence, so that everypolarization angle generates the same resonant reflection spectra, analternate biosensor structure can be used. Such an alternative biosensorcould consist of a plurality of concentric rings, such as the structureillustrated in FIG. 5(b). In this structure, the difference between aninside diameter and an outside diameter of each concentric ring is equalto about one-half of a grating period. Each successive ring has aninside diameter that is about one grating period greater than the insidediameter of the previous ring. The concentric ring pattern extends tocover a single sensor location—such as a microarray spot or a microtiterplate well. Each separate microarray spot or microtiter plate well has aseparate concentric ring pattern centered within it.

[0086] All polarization directions of such a structure have the samecross-sectional profile. The concentric ring structure, such as thestructure illustrated in FIG. 5(b), should be illuminated on-center topreserve polarization independence. The grating period of a concentricring structure is preferably less than the wavelength of the resonantlyreflected light. In one preferred embodiment, the grating period isabout 0.01 micron to about 1 micron and the grating depth is about 0.01to about 1 micron.

[0087] In another biosensor embodiment, an array of holes or posts areprovided on a sensor surface, such as the design illustrated in FIG.5(c). Preferably, the array of holes or posts are arranged toapproximate the concentric circle structure described above withoutrequiring the illumination beam to be centered upon any particularlocation of the grid. Such an array pattern is automatically generatedby the optical interference of three laser beams incident on a surfacefrom three directions at equal angles. In this pattern, the holes (orposts) are centered upon the corners of an array of closely packedhexagons, as illustrated in FIG. 5(c). The holes or posts also occur inthe center of each hexagon. Such a hexagonal grid of holes or posts hasthree polarization directions that “see” the same cross-sectionalprofile. The hexagonal grid structure, therefore, provides equivalentresonant reflection spectra using light of any polarization angle. Thus,no polarizing filter is required to remove unwanted reflected signalcomponents. The period of the holes or posts can be about 0.01 micronsto about 1 micron and the depth or height can be about 0.01 microns toabout 1 micron. These and various other grid structures are disclosedand described in commonly assigned co-pending patent application Ser.No. 09/930,352 herein entirely incorporated by reference and to whichthe reader is directed for further details.

[0088] One possible detection apparatus and method provides for resonantreflection structures and transmission filter structures comprisingconcentric circle gratings and hexagonal grids of holes or posts. For aresonant reflection structure, light output is measured on the same sideof the structure as the illuminating light beam. For example, asillustrated in FIG. 2, reflected light 29 is measured on the same sideof the structure 21 as the illuminating light beam or incident light 28.

[0089] For a transmission filter structure, light output is measured onthe opposite side of the structure as the illuminating beam. Thereflected and transmitted signals are complementary. That is, if awavelength is strongly reflected, it is weakly transmitted. Assuming noenergy is absorbed in the structure itself, the combined reflectedenergy and transmitted energy at a given wavelength will remain aconstant. The resonant reflection structure and transmission filters aredesigned to provide an efficient reflection at a specified wavelength.Therefore, a reflection filter will “pass” a narrow band of wavelengths,while a transmission filter will filter out or “cut” a narrow band ofwavelengths from incident light.

[0090] A resonant reflection structure or a transmission filterstructure can comprise a two-dimensional grating arranged in a patternof concentric circles. (See, e.g., FIG. 5(b). A resonant reflectionstructure or transmission filter structure can also comprise a hexagonalgrid of holes or posts. (See, e.g., FIG. 5(c)).

[0091] When these structures are illuminated with an illuminating lightbeam, a reflected radiation spectrum is produced. Such a radiationspectrum is independent of an illumination polarization angle of theilluminating light beam. A resonant grating effect is produced on thereflected radiation spectrum, wherein the depth and period of thetwo-dimensional grating or hexagonal grid of holes or posts are lessthan the wavelength of the resonant grating effect. These structuresreflect a narrow band of light when the structure is illuminated with abroadband of light.

[0092] Resonant reflection structures and transmission filter structuresof the invention can be used as biosensors. For example, one or morespecific binding substances can be immobilized on the hexagonal grid ofholes or posts or on the two-dimensional grating arranged in concentriccircles.

[0093] In an alternative embodiment, a reference resonant signal isprovided for more accurate measurement of peak resonant wavelengthshifts. The reference resonant signal can cancel out environmentaleffects, including, for example, temperature or other types of unwantednoise. A reference signal can be provided using a resonant reflectionsuperstructure that produces two separate resonant wavelengths. Forexample, FIG. 6A illustrates a transparent resonant reflectionsuperstructure arrangement 60. Transparent resonant reflectionsuperstructure 60 contains two sub-structures. The sub-structurescomprise a low n cured polymer 66 and a high n dielectric 67. A firstsub-structure comprises a first two-dimensional grating 62 with a top 61a and a bottom surface 61 b. The top surface of a two-dimensionalgrating 61 a comprises the grating surface 63. The first two-dimensionalgrating can comprise one or more specific binding substances immobilizedon its top surface. The top surface of the first two-dimensional grating63 will be in contact with a test sample (not shown). An optionalsubstrate layer 64 may provide support to the bottom surface 61 b of thefirst two-dimensional grating. The substrate layer 64 comprises a top 65a and bottom surface 65 b. The top surface 65 a of the substrate 64 isin contact with, and supports the bottom surface 61 b of the firsttwo-dimensional grating 62. A low n substrate 68 is also provided.

[0094] A second sub-structure 70, illustrated in FIG. 6B, comprises asecond two-dimensional grating 72 with a top surface 73 a and a bottomsurface 73 b. The second two-dimensional grating is not in contact witha test sample. The sub-structures comprise a low n cured polymer 77 anda high n dielectric 71. The second two-dimensional grating can befabricated onto a bottom surface of the substrate 74 that supports afirst two-dimensional grating 76. Where the second two-dimensionalgrating is fabricated on the substrate that supports the firsttwo-dimensional grating, the bottom surface 73 a of the secondtwo-dimensional grating 72 can be fabricated onto the bottom surface 75of the substrate 74. Therefore, the top surface 73 b of the secondtwo-dimensional grating 72 will face the opposite direction of a surface77 a of the first two-dimensional grating 76.

[0095] The top surface 73 b of the second two-dimensional grating 72 canalso be attached directly to the bottom surface of the firstsub-structure. In this arrangement, the top surface of the secondtwo-dimensional grating would face in the same direction as the topsurface 77 a of the first two-dimensional grating 76. A substrate 74 cansupport the bottom surface of the second two-dimensional grating in thisarrangement.

[0096] Because the second sub-structure is not in physical contact withthe test sample, its peak resonant wavelength is not subject to changesin the optical density of the test media, or deposition of specificbinding substances or binding partners on the surface of the firsttwo-dimensional grating. Therefore, such a superstructure produces tworesonant signals. Because the location of the peak resonant wavelengthin the second sub-structure is fixed, the difference in peak resonantwavelength between the two sub-structures provides a relative means fordetermining the amount of specific binding substances or bindingpartners or both deposited on the top surface of the first substructurethat is exposed to the test sample.

[0097] A biosensor superstructure can be illuminated from its topsurface or from its bottom surface, or from both surfaces. The peakresonance reflection wavelength of the first substructure is dependenton the optical density of material in contact with the superstructuresurface. The peak resonance reflection wavelength of the secondsubstructure is independent of the optical density of material incontact with the superstructure surface.

[0098] In an alternative embodiment, a biosensor is illuminated from abottom surface. Approximately 50% of the incident light is reflectedfrom the bottom surface of biosensor without reaching the active (or thetop) surface of the biosensor. A thin film or physical structure can beincluded in a biosensor composition that is capable of maximizing theamount of light that is transmitted to the upper surface of thebiosensor while minimizing the reflected energy at the resonantwavelength. The anti-reflection thin film or physical structure of thebottom surface of the biosensor can comprise, for example, a singledielectric thin film, or a stack of multiple dielectric thin films.

[0099] Alternatively, a “motheye” structure embossed into the bottombiosensor surface is provided. An example of a motheye structure isdisclosed in Hobbs, et al. “Automated interference lithography systemfor generation of sub-micron feature size patterns,” Proc. 1999Micromachine Technology for Diffracting and Holographic Optics, Societyof Photo-Optical Instrumentation Engineers, p. 124-135, (1999).

[0100] In one embodiment of the present invention, an interaction of afirst molecule with a second test molecule is detected. A SWS biosensoras previously described is used. However, there are no specific bindingsubstances immobilized on a SWS biosensor. Therefore, the biosensorcomprises a two-dimensional grating, a substrate layer that supports thetwo-dimensional grating. Optionally, a cover layer may be provided. Asdescribed above, when the biosensor is illuminated a resonant gratingeffect is produced on the reflected radiation spectrum, and the depthand period of the two-dimensional grating are less than the wavelengthof the resonant grating effect.

[0101] To detect an interaction of a first molecule with a second testmolecule, a mixture of the first and second molecules is applied to adistinct location on a biosensor. Such a location may be one area, spot,or one well on the biosensor. Alternatively, it could be a large area onthe biosensor. A mixture of the first molecule with a third controlmolecule is also applied to a distinct location on a biosensor. Thebiosensor can be the same biosensor as described above, or can be asecond biosensor. If the biosensor is the same biosensor, a seconddistinct location can be used for the mixture of the first molecule andthe third control molecule.

[0102] Alternatively, the same distinct biosensor location can be usedafter the first and second molecules are washed from the biosensor. Thethird control molecule does not interact with the first molecule and maybe about the same size as the first molecule. A shift in the reflectedwavelength of light from the distinct locations of the biosensor orbiosensors is measured using a read out method and apparatus as detailedbelow.

[0103] If the shift in the reflected wavelength of light from thedistinct location having the first molecule and the second test moleculeis greater than the shift in the reflected wavelength from the distinctlocation having the first molecule and the third control molecule, thenthe first molecule and the second test molecule interact. Interactioncan be, for example, hybridization of nucleic acid molecules, specificbinding of an antibody or antibody fragment to an antigen, and bindingof polypeptides. A first molecule, second test molecule, or thirdcontrol molecule can be, for example, a nucleic acid, polypeptide,antigen, polyclonal antibody, monoclonal antibody, single chain antibody(scFv), F(ab) fragment, F(ab′)₂ fragment, Fv fragment, small organicmolecule, cell, virus, and bacteria.

[0104] 3. Specific Binding Substances and Binding Partners

[0105] One or more specific binding substances may be immobilized on thetwo-dimensional grating or cover layer, if present. Immobilization mayoccur by physical adsorption or by chemical binding. A specific bindingsubstance can be, for example, a nucleic acid, polypeptide, antigen,polyclonal antibody, monoclonal antibody, single chain antibody (scFv),F(ab) fragment, F(ab′)₂ fragment, Fv fragment, small organic molecule,cell, virus, bacteria, or biological sample. A biological sample can befor example, blood, plasma, serum, gastrointestinal secretions,homogenates of tissues or tumors, synovial fluid, feces, saliva, sputum,cyst fluid, amniotic fluid, cerebrospinal fluid, peritoneal fluid, lunglavage fluid, semen, lymphatic fluid, tears, or prostatitc fluid.

[0106] Preferably, one or more specific binding substances are arrangedin a microarray of distinct locations on a biosensor. A microarray ofspecific binding substances comprises one or more specific bindingsubstances on a surface of a biosensor such that a biosensor surfacecontains a plurality of distinct locations, each with a differentspecific binding substance or with a different amount of a specificbinding substance. For example, an array can comprise 1, 10, 100, 1,000,10,000, or 100,000 distinct locations. A biosensor surface with a largenumber of distinct locations is called a microarray because one or morespecific binding substances are typically laid out in a regular gridpattern in x-y coordinates. However, a microarray can comprise one ormore specific binding substances laid out in a regular or irregularpattern. For example, distinct locations can define a microarray ofspots of one or more specific binding substances.

[0107] A microarray spot can range from about 50 to about 500 microns indiameter. Alternatively, a microarray spot can range from about 150 toabout 200 microns in diameter. One or more specific binding substancescan be bound to their specific binding partners.

[0108] In one biosensor embodiment, a microarray on a biosensor iscreated by placing microdroplets of one or more specific bindingsubstances onto, for example, an x-y grid of locations on atwo-dimensional grating or cover layer surface. When the biosensor isexposed to a test sample comprising one or more binding partners, thebinding partners will be preferentially attracted to distinct locationson the microarray that comprise specific binding substances that havehigh affinity for the binding partners. Some of the distinct locationswill gather binding partners onto their surface, while other locationswill not.

[0109] A specific binding substance specifically binds to a bindingpartner that is added to the surface of a biosensor of the invention. Aspecific binding substance specifically binds to its binding partner,but does not substantially bind other binding partners added to thesurface of a biosensor. For example, where the specific bindingsubstance is an antibody and its binding partner is a particularantigen, the antibody specifically binds to the particular antigen, butdoes not substantially bind other antigens. A binding partner can be,for example, a nucleic acid, polypeptide, antigen, polyclonal antibody,monoclonal antibody, single chain antibody (scFv), F(ab) fragment,F(ab′)₂ fragment, Fv fragment, small organic molecule, cell, virus,bacteria, and biological sample. A biological sample can be, forexample, blood, plasma, serum, gastrointestinal secretions, homogenatesof tissues or tumors, synovial fluid, feces, saliva, sputum, cyst fluid,amniotic fluid, cerebrospinal fluid, peritoneal fluid, lung lavagefluid, semen, lymphatic fluid, tears, and prostatitc fluid.

[0110] In an alternative embodiment, a nucleic acid microarray isprovided, in which each distinct location within the array contains adifferent nucleic acid molecule. In this embodiment, the spots withinthe nucleic acid microarray detect complementary chemical binding withan opposing strand of a nucleic acid in a test sample.

[0111] While microtiter plates are a common format used for biochemicalassays, microarrays are increasingly seen as a means for increasing thenumber of biochemical interactions that can be measured at one timewhile minimizing the volume of precious reagents. By application ofspecific binding substances with a microarray spotter onto a biosensorof the invention, specific binding substance densities of 10,000specific binding substances/in² can be obtained. By focusing anillumination beam of a fiber optic probe to interrogate a singlemicroarray location, a biosensor can be used as a label-free microarrayreadout system.

[0112] 4. Immobilization of One or More Specific Binding Substances

[0113] Immobilization of one or more binding substances onto a biosensoris performed so that a specific binding substance will not be washedaway by rinsing procedures, and so that its binding to binding partnersin a test sample is unimpeded by the biosensor surface. Severaldifferent types of surface chemistry strategies have been implementedfor covalent attachment of specific binding substances to, for example,glass for use in various types of microarrays and biosensors. Thesemethods can be adapted to a biosensor embodiment. Surface preparation ofa biosensor so that it contains certain required functional groups forbinding one or more specific binding substances can be an integral partof the biosensor manufacturing process.

[0114] One or more specific binding substances can be attached to abiosensor surface by physical adsorption (i.e., without the use ofchemical linkers). Alternatively, one or more specific bindingsubstances can be attached to a biosensor surface by chemical binding(i.e., with the use of chemical linkers). Chemical binding can generatestronger attachment of specific binding substances on a biosensorsurface. Chemical binding also provides defined orientation andconformation of the surface-bound molecules. Several examples ofchemical binding of specific binding substances to a biosensor embodyingone aspect of the invention are described in detail in related commonlyassigned co-pending patent application Ser. No. 09/930,352 hereinentirely incorporated by reference and to which the reader is directedfor further detail.

[0115] Other types of chemical binding include, for example, amineactivation, aldehyde activation, and nickel activation. These surfacescan be used to attach several different types of chemical linkers to abiosensor surface. While an amine surface can be used to attach severaltypes of linker molecules, an aldehyde surface can be used to bindproteins directly, without an additional linker. A nickel surface can beused to bind molecules that have an incorporated histidine (“his”) tag.Detection of “his-tagged” molecules with a nickel-activated surface iswell known in the art (Whitesides, Anal. Chem. 68, 490, (1996)).

[0116] Immobilization of specific binding substances to plastic, epoxy,or high refractive index material can be performed essentially asdescribed for immobilization to glass. However, an acid wash step may beeliminated where such a treatment would damage the material to which thespecific binding substances are immobilized.

[0117] For the detection of binding partners at concentrations of lessthan about ˜0.1 ng/ml, one may amplify and transduce binding partnersbound to a biosensor into an additional layer on the biosensor surface.The increased mass deposited on the biosensor can be detected as aconsequence of increased optical path length. By incorporating greatermass onto a biosensor surface, an optical density of binding partners onthe surface is also increased, thus rendering a greater resonantwavelength shift than would occur without the added mass. The additionof mass can be accomplished, for example, enzymatically, through a“sandwich” assay, or by direct application of mass to the biosensorsurface in the form of appropriately conjugated beads or polymers ofvarious size and composition. This principle has been exploited forother types of optical biosensors to demonstrate sensitivity increasesover 1500× beyond sensitivity limits achieved without massamplification. See, e.g., Jenison et al., “Interference-based detectionof nucleic acid targets on optically coated silicon,” NatureBiotechnology, 19: 62-65, 2001.

[0118] 5. Surface-Relief Volume Diffractive Biosensors

[0119] In an alternative embodiment, a biosensor comprises volumesurface-relief volume diffractive structures (a SRVD biosensor). SRVDbiosensors have a surface that reflects predominantly at a particularnarrow band of optical wavelengths when illuminated with a broad band ofoptical wavelengths. Where specific binding substances and/or bindingpartners are immobilized on a SRVD biosensor, the reflected wavelengthof light is shifted. One-dimensional surfaces, such as thin filminterference filters and Bragg reflectors, can select a narrow range ofreflected or transmitted wavelengths from a broadband excitation source.However, the deposition of additional material, such as specific bindingsubstances and/or binding partners onto their upper surface results onlyin a change in the resonance linewidth, rather than the resonancewavelength. In contrast, SRVD biosensors have the ability to alter thereflected wavelength with the addition of material, such as specificbinding substances and/or binding partners to the surface.

[0120] A SRVD biosensor comprises a sheet material having a first andsecond surface. The first surface of the sheet material defines reliefvolume diffraction structures. Sheet material can comprise, for example,plastic, glass, semiconductor wafer, or metal film.

[0121] A relief volume diffractive structure can be, for example, atwo-dimensional grating, as described above, or a three-dimensionalsurface-relief volume diffractive grating. The depth and period ofrelief volume diffraction structures are less than the resonancewavelength of light reflected from a biosensor.

[0122] A three-dimensional surface-relief volume diffractive grating canbe, for example, a three-dimensional phase-quantized terraced surfacerelief pattern whose groove pattern resembles a stepped pyramid. Whensuch a grating is illuminated by a beam of broadband radiation, lightwill be coherently reflected from the equally spaced terraces at awavelength given by twice the step spacing times the index of refractionof the surrounding medium. Light of a given wavelength is resonantlydiffracted or reflected from the steps that are a half-wavelength apart,and with a bandwidth that is inversely proportional to the number ofsteps. The reflected or diffracted color can be controlled by thedeposition of a dielectric layer so that a new wavelength is selected,depending on the index of refraction of the coating.

[0123] A stepped-phase structure can be produced first in photoresist bycoherently exposing a thin photoresist film to three laser beams, asdescribed previously. See e.g., Cowen, “The recording and large scalereplication of crossed holographic grating arrays using multiple beaminterferometry,” in International Conference on the Application, Theory,and Fabrication of Periodic Structures, Diffraction Gratings, and MoirePhenomena II, Lerner, ed., Proc. Soc. Photo-Opt. Instrum. Eng., 503,120-129, 1984; Cowen, “Holographic honeycomb microlens,” Opt. Eng. 24,796-802 (1985); Cowen & Slafer, “The recording and replication ofholographic micropatterns for the ordering of photographic emulsiongrains in film systems,” J. Imaging Sci. 31, 100-107, 1987. Thenonlinear etching characteristics of photoresist are used to develop theexposed film to create a three-dimensional relief pattern. Thephotoresist structure is then replicated using standard embossingprocedures. For example, a thin silver film may be deposited over thephotoresist structure to form a conducting layer upon which a thick filmof nickel can be electroplated. The nickel “master” plate is then usedto emboss directly into a plastic film, such as vinyl, that has beensoftened by heating or solvent.

[0124] A theory describing the design and fabrication ofthree-dimensional phase-quantized terraced surface relief pattern thatresemble stepped pyramids is described: Cowen, “Aztec surface-reliefvolume diffractive structure,” J. Opt. Soc. Am. A, 7:1529 (1990).

[0125] An example of a three-dimensional phase-quantized terracedsurface relief pattern may be a pattern that resembles a steppedpyramid. Each inverted pyramid is approximately 1 micron in diameter.Preferably, each inverted pyramid can be about 0.5 to about 5 micronsdiameter, including for example, about 1 micron. The pyramid structurescan be close-packed so that a typical microarray spot with a diameter of150-200 microns can incorporate several hundred stepped pyramidstructures. The relief volume diffraction structures have a period ofabout 0.1 to about 1 micron and a depth of about 0.1 to about 1 micron.

[0126]FIG. 6 illustrates a reflective surface 82 of a biosensor 80. Thereflective surface 82 includes a first microarray spot 84 and a secondmicroarray spot 86. The first microarray spot 82 is not provided with anadsorbed material while the second microarray spot 84 is provided withan adsorbed protein. Consequently, the index or refraction for the firstspot remains unchanged, i.e., n_(air)=1. The index of refraction for thesecond spot will change to that of an absorbed protein, i.e.,n_(protein)=1.4.

[0127] As white light 81 is illuminated onto the first microarray spot84, blue light 89 will be reflected. Alternatively, the reflected lightof white light 88 illuminated unto the second microarray spot 86 will bedifferent (i.e., green light 87) because of the different index ofrefraction.

[0128]FIG. 6 demonstrates how individual microarray locations (with anentire microarray spot incorporating hundreds of pyramids nowrepresented by a single pyramid for one microarray spot) can beoptically queried to determine if specific binding substances or bindingpartners are adsorbed onto the surface. When the structure isilluminated with white light, structures without significant boundmaterial will reflect wavelengths determined by the step height of thestructure. When higher refractive index material, such as bindingpartners or specific binding substances, are incorporated over thereflective metal surface, the reflected wavelength is modified to shifttoward longer wavelengths. The color that is reflected from the terracedstep structure is theoretically given as twice the step height times theindex of refraction of a reflective material that is coated onto thefirst surface of a sheet material of a SRVD biosensor. A reflectivematerial can be, for example silver, aluminum, or gold.

[0129] One or more specific binding substances, as described above, areimmobilized on the reflective material of a SRVD biosensor. One or morespecific binding substances can be arranged in microarray of distinctlocations, as described above, on the reflective material. FIG. 7illustrates an embodiment of a microarray sensor 90. In this embodiment,the microarray biosensor 90 comprises a 9-element microarray biosensor.A plurality of individual grating structures, represented in FIG. 7 bysmall circles, such as small circle 91, lie within each microarray spot.The microarray spots, represented by the larger circles 92(a-i), reflectwhite light at a specific wavelength. This specific wavelength isdetermined by the refractive index of material on the microarraysurface. Microarray locations with additional adsorbed material willhave reflected wavelengths that are shifted toward longer wavelengths,represented by the larger circles.

[0130] Because the reflected wavelength of light from a SRVD biosensoris confined to a narrow bandwidth, very small changes in the opticalcharacteristics of the surface manifest themselves in easily observedchanges in reflected wavelength spectra. The narrow reflection bandwidthprovides a surface adsorption sensitivity advantage compared toreflectance spectrometry on a flat surface.

[0131] A SRVD biosensor reflects light predominantly at a first singleoptical wavelength when illuminated with a broad band of opticalwavelengths, and reflects light at a second single optical wavelengthwhen one or more specific binding substances are immobilized on thereflective surface. The reflection at the second optical wavelengthresults from optical interference. A SRVD biosensor also reflects lightat a third single optical wavelength when the one or more specificbinding substances are bound to their respective binding partners, dueto optical interference.

[0132] Readout of the reflected color can be performed serially byfocusing a microscope objective onto individual microarray spots andreading the reflected spectrum with the aid of a spectrograph or imagingspectrometer, or in parallel by, for example, projecting the reflectedimage of the microarray onto an imaging spectrometer incorporating ahigh resolution color CCD camera.

[0133] A SRVD biosensor can be manufactured by, for example, producing ametal master plate, and stamping a relief volume diffractive structureinto, for example, a plastic material like vinyl. After stamping, thesurface is made reflective by blanket deposition of, for example, a thinmetal film such as gold, silver, or aluminum. Compared to MEMS-basedbiosensors that rely upon photolithography, etching, and wafer bondingprocedures, the manufacture of a SRVD biosensor is very inexpensive.

[0134] 6. Liquid-Containing Vessels

[0135] A SWS or SRVD biosensor embodiment can comprise an inner surface.In one preferred embodiment, such an inner surface is a bottom surfaceof a liquid-containing vessel. A liquid-containing vessel can be, forexample, a microtiter plate well, a test tube, a petri dish, or amicrofluidic channel. In one embodiment, a SWS or SRVD biosensor isincorporated into a microtiter plate.

[0136] For example, a SWS biosensor or SRVD biosensor can beincorporated into the bottom surface of a microtiter plate by assemblingthe walls of the reaction vessels over the resonant reflection surface,so that each reaction “spot” can be exposed to a distinct test sample.Therefore, each individual microtiter plate well can act as a separatereaction vessel. Separate chemical reactions can, therefore, occurwithin adjacent wells without intermixing reaction fluids and chemicallydistinct test solutions can be applied to individual wells.

[0137] Several methods for attaching a biosensor of the invention to thebottom surface of bottomless microtiter plates can be used, including,for example, adhesive attachment, ultrasonic welding, and laser welding.

[0138] The most common assay formats for pharmaceutical screeninglaboratories, molecular biology research laboratories, and diagnosticassay laboratories are microtiter plates. The plates are standard-sizedplastic cartridges that can contain 96, 384, or 1536 individual reactionvessels arranged in a grid. Due to the standard mechanical configurationof these plates, liquid dispensing, robotic plate handling, anddetection systems are designed to work with this common format. Abiosensor incorporating an embodiment of the present invention can beincorporated into the bottom surface of a standard microtiter plate.See, e g., FIGS. 8A & 8B.

[0139] For example, FIG. 8A illustrates a microtiter plate 93. Themicrotiter plate 93 is a bottomless microtiter plate having a pluralityof holes 93(a). The plurality of holes, preferably arranged in an array,extend from a top surface 96 to a bottom surface 95 of the microtiterplate 93. A microarray slide 94 (FIG. 8B) is provided along the platebottom surface 95. The slide acts as a resonant reflection biosensorsurface due to the incorporation of structure in the bottom surface inaccordance with FIGS. 4 or 5, described previously.

[0140] Because the biosensor surface can be fabricated in large areas,and because a readout system incorporating one aspect of the presentinvention does not make physical contact with the biosensor surface, aplurality of individual biosensor areas can be defined.

[0141] 7. Holding Fixtures

[0142] A number of biosensors that are, for example, about 1 mm² toabout 5 mm², and preferably less than about 3×3 mm² can be arranged ontoa holding fixture that can simultaneously dip the biosensors intoseparate liquid-containing vessels, such as wells of a microtiter plate,for example, a 96-, 384-, or 1536-well microtiter plate. Other types ofliquid containing vessels could also be used including a micro fluidicdevice, a microarray chip, a petri dish, a microscope slide, and aflask.

[0143]FIG. 9 illustrates an embodiment of a holding fixture 100. Fixture100 includes a plurality of wells 104. In this embodiment, the fixtureincludes 96 wells arranged in an array comprising 8 rows and 12 columns.Each well 104 includes a plurality of microtiter plate spots 102. Where,for example, the biosensor includes 96 wells, a total of 4800 platespots are provided. (4800=96×50). Therefore, with the embodimentillustrated in FIG. 9, this holding fixture 100 could be dipped into a96-well plate and perform 4800 assays.

[0144] Each of the biosensors can contain multiple distinct locations. Aholding fixture has one or more attached biosensors so that eachindividual biosensor can be lowered into a separate, liquid-containingvessel. A holding fixture can comprise plastic, epoxy or metal. Forexample, 50, 96, 384, or 1,000, or 1,536 biosensors can be arranged on aholding fixture, where each biosensor has 25, 100, 500, or 1,000distinct locations. As an example, where 96 biosensors are attached to aholding fixture and each biosensor comprises 100 distinct locations,9600 biochemical assays can be simultaneously performed.

[0145] 8. Methods of using SWS and SRVD Biosensors

[0146] The disclosed SWS and SRVD biosensors can be used to study one ora number of specific binding substance/binding partner interactions inparallel. Binding of one or more specific binding substances to theirrespective binding partners can be detected, without the use of labels.Detection occurs by applying one or more binding partners to a SWS orSRVD biosensor that have one or more specific binding substancesimmobilized on their surfaces. A SWS biosensor is illuminated with lightand a maxima in reflected wavelength, alternatively or a minima intransmitted wavelength is detected from the biosensor. If one or morespecific binding substances have bound to their respective bindingpartners, then the reflected wavelength of light is shifted as comparedto a situation where one or more specific binding substances have notbound to their respective binding partners. The shift is detected by aspectrographic device as described in further detail. Where a SWSbiosensor is coated with an array of distinct locations containing theone or more specific binding substances, then a maxima in reflectedwavelength or minima in transmitted wavelength of light is detected fromeach distinct location of the biosensor.

[0147] A SRVD biosensor is illuminated with light after binding partnershave been added and the reflected wavelength of light is detected fromthe biosensor. Where one or more specific binding substances have boundto their respective binding partners, the reflected wavelength of lightis shifted.

[0148] In an alternative embodiment, a variety of specific bindingsubstances, for example, antibodies, can be immobilized in an arrayformat onto a biosensor of the invention. The biosensor is contactedwith a test sample of interest comprising binding partners, such asproteins. Only the proteins that specifically bind to the antibodiesimmobilized on the biosensor remain bound to the biosensor. Such anapproach is essentially a large-scale version of an enzyme-linkedimmunosorbent assay. However, in this embodiment, the use of an enzymeor fluorescent label is not required.

[0149] The activity of an enzyme can be detected by applying one or moreenzymes to a SWS or SRVD biosensor to which one or more specific bindingsubstances have been immobilized. The biosensor is washed andilluminated with light. The reflected wavelength of light is detectedfrom the biosensor. Where the one or more enzymes have altered the oneor more specific binding substances of the biosensor by enzymaticactivity, the reflected wavelength of light is shifted.

[0150] Additionally, a test sample, for example, cell lysates containingbinding partners can be applied to a biosensor of the invention,followed by washing to remove unbound material. The binding partnersthat bind to a biosensor can be eluted from the biosensor and identifiedby, for example, mass spectrometry. Optionally, a phage DNA displaylibrary can be applied to a biosensor of the invention followed bywashing to remove unbound material. Individual phage particles bound tothe biosensor can be isolated and the inserts in these phage particlescan then be sequenced to determine the identity of the binding partner.

[0151] For the above applications, and in particular proteomicsapplications, the ability to selectively bind material, such as bindingpartners from a test sample onto a preferred biosensor, followed by theability to selectively remove bound material from a distinct location ofthe biosensor for further analysis is advantageous. Biosensors may alsobe capable of detecting and quantifying the amount of a binding partnerfrom a sample that is bound to a biosensor array distinct location bymeasuring the shift in reflected wavelength of light. For example, thewavelength shift at one distinct biosensor location can be compared topositive and negative controls at other distinct biosensor locations todetermine the amount of a binding partner that is bound to a biosensorarray distinct location.

[0152] 9. SWS and Electrically Conducting Material

[0153] An alternative biosensor embodiment structure is provided thatenables a biosensor array to selectively attract or repel bindingpartners from individual distinct locations on a biosensor. As is wellknown in the art, an electromotive force can be applied to biologicalmolecules such as nucleic acids and amino acids subjecting them to anelectric field. Because these molecules are electronegative, they areattracted to a positively charged electrode and repelled by a negativelycharged electrode.

[0154] In one embodiment of the present invention, a grating structureof a resonant optical biosensor is provided with an electricallyconducting material rather than an electrically insulating material. Anelectric field is applied near the biosensor surface. Where a gratingoperates as both a resonant reflector biosensor and as an electrode, thegrating comprises a material that is both optically transparent near theresonant wavelength, and also has low resistivity. In one biosensorembodiment, the material is indium tin oxide, InSn_(x)O_(1−x) (ITO). ITOis commonly used to produce transparent electrodes for flat paneloptical displays, and is therefore readily available at low cost onlarge glass sheets. The refractive index of ITO can be adjusted bycontrolling x, the fraction of Sn that is present in the material.Because the liquid test sample solution has mobile ions (and willtherefore be an electrical conductor), the ITO electrodes are coatedwith an insulating material. In a preferred resonant optical biosensorembodiment, a grating layer is coated with a layer with lower refractiveindex material. Materials such as cured photoresist (n=1.65), curedoptical epoxy (n=1.5), and glass (n=1.4-1.5) are strong electricalinsulators that also have a refractive index that is lower than ITO(n=2.0-2.65). A cross-sectional diagram of a biosensor that incorporatesan ITO grating is shown in FIG. 16.

[0155] As illustrated in FIG. 16, n₁ represents the refractive index ofan electrical insulator 222. n₂ represents the refractive index of atwo-dimensional grating 224. t₁ represents the thickness of theelectrical insulator 222. t₂ represents the thickness of thetwo-dimensional grating 224. n_(b10) represents the refractive index ofone or more specific binding substances 228 and t_(B10) represents thethickness of the one or more specific binding substances 228.

[0156] A grating can be a continuous sheet of ITO contains an array ofregularly spaced holes. The holes are filled in with an electricallyinsulating material, such as cured photoresist. The electricallyinsulating layer overcoats the ITO grating so that the upper surface ofthe structure is completely covered with electrical insulator, and sothat the upper surface is substantially flat. When the biosensor 220 isilluminated with light 230, a resonant grating effect is produced on thereflected radiation spectrum 225. The depth and the period of thegrating 224 are less than the wavelength of the resonant grating effect.

[0157] A single electrode can comprise a region containing a pluralityof grating periods. For example, FIG. 10 illustrates an embodiment of anarray of biosensor electrodes 110. In this preferred embodiment, thearray of biosensor electrodes 110 includes 9 grating periods 112(a-i).Providing a plurality of separate grating regions 112(a-i) on a singlesubstrate surface creates an array of biosensor electrodes. Electricalcontact to regions 112(a-i) is provided using electrically conductingtraces 114(a-i). Traces 114(a-i) are preferably constructed from thesame material as a conductor within the biosensor electrode 110.Conducting traces 114(a-i) are coupled to a voltage source 115. Voltagesource 115 applies an electrical potential to the various gratingregions.

[0158]FIG. 11 illustrates a SEM photograph 116 illustrating a pluralityof separate grating regions 117(a-f) of an array of biosensorelectrodes, such as the electrode illustrated in FIG. 10.

[0159] To apply an electrical potential to a biosensor that is capableof attracting or repelling a molecule near the electrode surface, abiosensor upper surface can be immersed in a liquid sample.

[0160]FIG. 12(a) illustrates an embodiment of a biosensor upper surfaceimmersed in a liquid sample 130. The biosensor includes a substrate 124,an ITO rating 125, and an insulator 126. A “common” electrode 122 can beplaced within the sample liquid 130. A voltage from source 123 can beapplied between one selected biosensor electrode region 125 and thecommon electrode. In this manner, one, several, or all electrodes may beactivated or inactivated simultaneously. FIG. 12(b) illustrates theattraction of electronegative molecules 144 to the biosensor surfacewhen a positive voltage is applied to the electrode. FIG. 12(c)illustrates the application of a repelling force such as a reversedelectrical charge to electronegative molecules 144 using a negativeelectrode voltage 123(b).

[0161] 10. Detection Apparatus and System

[0162]FIG. 13 illustrates one embodiment of a detection system 150. Thedetection system 150 comprises a biosensor 152 having the structure aspreviously herein described, e.g., as in FIG. 4. The detection system150 further includes a light source 154 and spectrometer 160. The lightsource 154 directs light to the biosensor 152. A detector, such as thespectrometer 160, detects light reflected via a collecting fiber 153from the biosensor.

[0163] The light source 154 illuminates the biosensor 151 via anilluminating fiber 152 from a sensor top surface, i.e., the surface towhich one or more specific binding substances are immobilized.Alternatively, the biosensor 152 may be illuminated from its bottomsurface. By measuring the shift in resonant wavelength at each distinctlocation of the biosensor 152, it is possible to determine whichdistinct locations have binding partners bound to them. The extent ofthe shift can be used to determine the amount of binding partners in atest sample and the chemical affinity between one or more specificbinding substances and the binding partners of the test sample.

[0164] In one embodiment, the biosensor 152 is illuminated twice. Afirst reflected measurement determines the reflectance spectra of one ormore distinct locations of a biosensor array with one or more specificbinding substances immobilized on the biosensor. A second measurementdetermines the reflectance spectra after one or more binding partnersare applied to a biosensor. The difference in peak wavelength betweenthese two measurements is a measurement of the amount of bindingpartners that have specifically bound to a biosensor or one or moredistinct locations of a biosensor. This method of illumination canaccount for small nonuniformities in a surface of a biosensor, resultingin regions with slight variations in the peak resonant wavelength. Thismethod can detect varying concentrations or molecular weights ofspecific binding substances immobilized on a biosensor.

[0165] Computer simulation can be used to determine the expecteddependence between a peak resonance wavelength and an angle of incidentillumination. For example, referring to FIG. 1, substrate 12 may bechosen as glass (n_(substrate)=1.50). The grating 16 could be atwo-dimensional pattern of silicon nitride squares (t₂=180 nm, n₂=2.01(n=refractive index), k₂=0.001 (k=absorption coefficient)) with a periodof 510 nm, and a filling factor of 56.2% (i.e., 56.2% of the surface iscovered with silicon nitride squares while the rest is the area betweenthe squares). The areas between silicon nitride squares may be filledwith a lower refractive index material. The same material covers thesquares and provides a uniformly flat upper surface. In this embodiment,a glass layer may be selected (n₁=1.40) such that it covers the siliconnitride squares by t₂=100 nm.

[0166] The reflected intensity as a function of wavelength was modeledusing GSOLVER software. Such software utilizes full 3-dimensional vectorcode using hybrid Rigorous Coupled Wave Analysis and Modal analysis.GSOLVER calculates diffracted fields and diffraction efficiencies fromplane wave illumination of arbitrarily complex grating structures. Theillumination can be from any incidence and any polarization.

[0167]FIG. 14 illustrates a graphical representation 164 of a resonancewavelength of a biosensor measured as a function of incident angle of anillumination beam. The simulation demonstrates that there is acorrelation between the angle of incident light, and the measured peakwavelength. This result implies that the collimation of the illuminatingbeam, and the alignment between the illuminating beam and the reflectedbeam affects the measured resonant peak linewidth. If the collimation ofthe illuminating beam is poor, a range of illuminating angles will beincident on the biosensor surface, and a wider resonant peak will bemeasured than if purely collimated light were incident.

[0168] Because the lower sensitivity limit of a biosensor is related tothe ability to determine a peak maxima, it is important to measure anarrow resonant peak. Therefore, the use of a collimating illuminationsystem provides increased sensitivity.

[0169] It may be desirable for the illuminating and collecting fiberprobes to spatially share the same optical path. Several methods may beused to co-locate illuminating and collecting optical paths. Forexample, one method includes a single illuminating fiber, which may becoupled at its first end to a light source that directs light at thebiosensor, and a single collecting fiber, which may be coupled at itsfirst end to a detector that detects light reflected from the biosensor.Each can be coupled at their second ends to a third fiber probe suchthat this third fiber acts as both an illuminator and a collector. Thethird fiber probe is oriented at a normal angle of incidence to thebiosensor and supports counter-propagating illuminating and reflectingoptical signals.

[0170]FIG. 15 illustrates an alternative embodiment of a detectionsystem 170. In this alternative embodiment, the detection system 170includes a beam splitter 174. The beam splitter 174 enables a singleilluminating fiber 178, which is optically coupled to a light source, tobe oriented at a 90 degree angle to a collecting fiber 176. Thecollecting fiber 176 is optically coupled to a detector. Light isdirected through the illuminating fiber probe into the beam splitter,which directs light toward the biosensor 172. The reflected light isdirected back into the beam splitter 174, which then directs reflectedlight into the collecting fiber probe 176.

[0171] A beam splitter allows the illuminating light and the reflectedlight to share a common optical path between the beam splitter and thebiosensor.

[0172]FIG. 17 illustrates an optical fiber probe measuring apparatus202. FIG. 17 is a basic design for a PWV detector that can be adapted toa variety of possible instrumentation configurations. Generally, themeasuring apparatus 202 includes an instrument that detects biochemicalinteractions occurring on a surface 215 of an optical biosensor 211. Aspreviously detailed above, the biosensor 211 may be embedded within abottom portion of a conventional microtiter plate. In one embodiment,the measuring apparatus 202 illuminates the biosensor surface 215 bycasting a spot of collimated white light onto the sensor structure 211.

[0173] The measuring apparatus 202 collects light reflected from theilluminated biosensor surface. Preferably, the apparatus illuminates andgathers light from multiple locations on the biosensor surfacesimultaneously. In another embodiment, the apparatus 202 scans thedetection head 209 of a dual illumination probe across the biosensorsurface. Based on the reflected light, the apparatus measures certainvalues, such as the peak wavelength values (PWV's), of a plurality oflocations within the biosensor embedded microtiter plate.

[0174] The biosensor can be incubated in a incubation enclosure andmoved to a position for reading. One possible configuration of aninstrument that performs incubation and reading is set forth below.Incubation may occur at a user determined temperature. An instrumentincorporating the measuring apparatus may also provide a mechanism formixing samples within a microtiter plate well while the optical sensorresides inside the apparatus. The mixing could take the form of ashaking mechanism or other type of system.

[0175] As illustrated in FIG. 17, measuring apparatus 202 includes awhite light source 205, an optical fiber probe 208, a collimating lens210, and a spectrometer 212. A liquid test sample is placed in the spacebetween the structures 216 and 217 for binding to receptors on thesurface 215. The measuring apparatus 202 measures biochemicalinteractions occurring on a surface 215 of the optical device 211.Optical device 211 may have the characteristics of the biosensor devicesas herein previously described, such as a well of microtiter platemodified in accordance with FIG. 8A and 8B. Advantageously, thesemeasurements occur without the use of fluorescent tags or colorimetriclabels.

[0176] As described in more detail above, the surface 215 of the opticaldevice 211 contains an optical structure such as shown in FIG. 17. Whenilluminated with collimated white light generated by light source 205,optical device surface 215 is designed to reflect only a narrow band ofwavelengths. This narrow band of wavelengths is described as awavelength peak. The “peak wavelength value” (i.e., PWV) changes whenbiological material is deposited or removed from the sensor surface 215.That is, the PWV changes when a biological material is deposited betweenthe structures 216 and 217.

[0177] The measuring apparatus 202 illuminates distinct locations on theoptical device surface with collimated white light, and then collectsthe reflected light. The collected light is gathered into a wavelengthspectrometer 212 for processing, including generation of a PWV.

[0178] The measuring apparatus 202 utilizes at least one optical fiberprobe 208. Referring now to both FIGS. 17 and 18(a), optical fiber probe208 can comprise both an illuminating fiber 218 and a detecting fiber220. The illuminating fiber 218 is optically coupled to the white lightsource 205 and terminates at a probe head 209 of the optical fiber probe208. Detecting fiber 220 is optically coupled to a spectrometer 212 andterminates at the probe head 209 of the optical fiber probe 208. In thisembodiment, the spectrometer 212 is a single-point spectrometer.

[0179] The light source generates white light. The illuminating fiber218 directs an area of collimated light, via the collimating lens 210,on a sensor surface, preferably a bottom sensor surface. Preferably, theilluminating fiber is bundled along with the second detecting fiber andcontained in a unitary optical fiber probe, such as fiber optic probe208. This detecting fiber 220 is utilized to collect light reflectedfrom the biosensor 211. The detecting fiber 220 channels the reflectedlight to the spectrometer unit 212, preferably a wavelengthspectrometer. The spectrometer unit 212 processes the reflected light todetermine a resonance peak wavelength value (i.e., PWV) of the reflectedlight.

[0180] In one preferred embodiment of a measuring apparatus, white lightsource 205 illuminates a ˜1 millimeter (mm) diameter region of thegrating region 215 through a 400 micrometer diameter fiber optic and thecollimating lens 210 at nominally normal incidence through the bottom ofa microtiter plate. Such a microtiter plate could have a standard 96-,384-, or 1526-well microtiter plate format, but with a biosensorattached to the bottom.

[0181] The measuring apparatus 202 illustrated in FIG. 17 includes asingle-point optical spectrometer included in the spectrometer unit 212.Preferably, the spectrometer unit 212 includes a commercially availableoptical spectrometer such as those spectrometers commercially availablefrom Ocean Optics, Inc. Spectrometer 212 includes a diffraction gratingand a linear or one dimensional charge coupled device (CCD). Thediffraction grating breaks the incident radiation into its componentspectra. The boundaries of the spectra may generally range from 680nanometers (nm) to 930 nm for the device.

[0182] The incident radiation impinges a linear (or one dimensional)CCD. In one embodiment, this one dimensional CCD has 2048 pixels. TheCCD converts incident radiation into an electric charge. There is ageneral relationship between the incident radiation and the resultingelectric charge: the greater the number of incident photons, the greaterthe amount of charge the pixels in the CCD accumulate.

[0183] Each pixel in the CCD images a separate wavelength of light dueto the spatial separation provided by the grating in the spectrometerand the distance between the grating and the CCD. The gap in wavelengthimaged by each pixel may range from about 0.13 to about 0.15 nm.Therefore, the first pixel in the CCD images light at a wavelength of680 nm, the second pixel at 680.13 nm, the third pixel at 680.27 nm,etc. The 2048th pixel, therefore, images light at approximately 930 nm.

[0184] The CCD readout consists of an analog voltage signal for eachpixel. These analog voltage signals are converted to a digital signal,ranging in value from 0 to 4,095. A digital value of 0 implies thatthere is no signal (i.e., there is no incident radiation). Conversely, adigital value of 4,095 implies that the pixel is saturated with incidentradiation.

[0185] A general arrangement of the spectrometer 212 of FIG. 7 isprovided in FIG. 18(b). For ease of explanation, the imaging opticsinside the spectrometer are not shown in FIG. 18(b). As illustrated inFIG. 18(b), light 221 is incident on the spectrographic grating 222.This light is then reflected in a dispersed fashion 223, 225 along asurface 224 of the CCD 226 with the separation of light in accordancewith the shorter wavelength λ₁ imaged at a first end of the CCD and thelonger wavelength λ₂ imaged at a second end of the CCD.

[0186]FIG. 18(c) illustrates a readout of the CCD (Y axis) as a functionof pixel number. The curve 229 indicates that there is a peak 231 inpixel output for a particular wavelength λ peak. λ peak is the peakwavelength value (PWV) referred to herein elsewhere.

[0187] Through calibration, one can determine the relationship betweenwavelength λ and the CCD pixel number, the pixel number beingrepresented in FIG. 18(c) along the x-axis. Commercially availablewavelength spectrometers, such as the spectrometers commerciallyavailable from Ocean Optics, Inc., are provided with informationdetailing a spectrometer's calibration.

[0188] For a given microtiter plate well illuminated by the measuringapparatus 202, the spectrometer provides a curve of CCD readout as afunction of λ or pixel number. Thus, an instrument will generate datasimilar to that shown in FIG. 18c and determine PWV for each well ordetection location on the sensor.

[0189] In an alternative embodiment, a measuring apparatus is providedthat simultaneously illuminates and measures a plurality of opticaldevice surface regions. In such an alternative embodiment, a pluralityof dual probe fibers are utilized. For example, FIG. 19 illustrates analternative embodiment of a measuring apparatus 260.

[0190] Measuring apparatus 260 includes a plurality of dual fiber probes262(a-h) for illuminating and detecting light reflected from opticaldevice surface. In this embodiment, the apparatus 260 includes eight (8)dual fiber probes designated generally as 262(a-h). However, as those ofordinary skill will realize, measuring apparatus 260 could includealternative dual fiber probe arrangements.

[0191] Preferably, dual probe fibers 262(a-h) function as bothilluminators and detectors for a single row of microtiter wells providedin bottomless microtiter plate 267. As illustrated, liquid 268 may beprovided in a first microtiter plate well 270. For a conventional96-well microtiter plate, there would be 8 microtiter wells 268(a-h) inthis row.

[0192] In one embodiment, the measuring apparatus 260 includes a singlespectrometer included in spectrometer unit 263 and a single light source261. Alternatively, measuring apparatus 260 could include a separatespectrometer and a separate CCD for each dual probe fiber: i.e., thereare 8 spectrometers and 8 CCDs included in the spectrometer unit 263.Alternatively, the measuring apparatus 260 could include a separatelight source for each dual probe in light source unit 261. Theprocessing, measurement, and readout of reflected light for each well issimilar to the processing and readout provided in FIG. 18(c).

[0193] The measurement system 260 illustrated in FIG. 19 includes aplurality of dual fiber probes 262. Each dual fiber probe operates in asimilar fashion as the dual probe illustrated in FIG. 17 and asdescribed above.

[0194] Preferably, an algorithm processing the curve illustrated in FIG.18(c) determines a peak wavelength value with sub-pixel resolution.Sub-pixel resolution is generally preferred since this type ofresolution provides peak wavelength values with a precision greater thanthe CCD pixel separation (i.e., 0.13 to 0.15 nm). A presently preferredalgorithm is set forth in section 12 below.

[0195] In the dual fiber probe embodiment illustrated in FIG. 19, eachdual probe 262 (a-h) includes an illuminating fiber and a detectingfiber. In this preferred embodiment, the apparatus 260 is configured toinclude eight dual fiber probes and therefore provides a separatereadout per dual probe. In such a configuration, eight dual fiber probessimultaneously illuminate a single row of a standard microtiter plateand measure light reflected from this illuminated row. As those ofordinary skill will realize, other multiple probe configurations mayalso be utilized.

[0196] The plurality of dual probes may be arranged side by side in alinear fashion. By utilizing such a linear arrangement, a plurality ofdual probes can simultaneously illuminate and then read out a pluralityof sensor surface locations. For example, a linear probe arrangement maybe utilized to illuminate and then read an entire row or an entirecolumn of a microtiter plate. In this preferred embodiment, each dualprobe head contains two optical fibers. The first fiber is connected toa white light source to cast a small spot of collimated light on thesensor surface. The second fiber reflects the reflected radiation andsupplies it to a spectrometer. After one row is illuminated, relativemotion occurs between the detector probes and the sensor (microtiterplate) and the next row or column of the sensor is read. The processcontinues until all rows (or columns) have been read.

[0197] As will be described in further detail below, in one embodimentof the measuring apparatus, a microtiter plate is placed on a linearmotion stage. The linear motion stage moves the microplate in aspecified, linear scan direction. As the microtiter plate is moved inthis scan direction, each microplate column is sequentially illuminated.The resulting reflected light is measured. In one preferred embodiment,a scan of a conventional 96-well microtiter plate may take approximately15 to 30 seconds to illuminate and measure the resultant reflectedspectrum.

[0198] In yet another alternative embodiment, an imaging apparatusutilizes a spectrometer unit that comprises an imaging spectrometer. Forexample, FIG. 20 illustrates an alternative embodiment of a biosensorinstrument system 319. The instrument system 319 includes an imagingspectrometer 332. One advantage of the imaging spectrometer system isthat such imaging systems reduce the amount of time for generating a PWVimage. Another advantage is to study biological binding of an area in anon-uniform fashion.

[0199] The instrument system 319 illustrated in FIG. 20 includes acollimated white light source 320, a beam splitter 324, and an imagingspectrometer 330. Beam splitter 324 redirects the collimated light 322towards a biosensor in accordance with principles discussed previously.In one embodiment, the biosensor 319 is a conventional microarray chip328. More preferably, the microarray chip could comprise a plurality ofwells or spots arranged in an array of uniform rows and columns.

[0200] Imaging spectrometer 332 is used to generate a PWV image for eachof the receptor locations (spots) contained in a microarray chip 328. Inthis embodiment, the microarray chip 328 comprises a plurality of spotwarranged in eight (8) rows, where each row contains six (6) spotw. Forexample, a first microarray chip row 331 contains six spotw 331(a-f).Similarly, a second microarray chip row 333 contains six spotw 333(a-f).All the spotw of each row can be simultaneously illuminated incidentlight 332. As those of ordinary skill in the art will recognize, thetotal number of spots on a microarray chip can be much larger, routinelyin the tens of thousands.

[0201] Light 322 is collimated and directed towards the beam splitter324. The beam splitter 324 allows the collimated light 322 and thereflected light 334 to share a common optical path between the beamsplitter and the microarray chip 328.

[0202] Preferably, the collimated light is directed at the sensor bottomsurface 342 at normal incidence via the beam splitter 324. Normallyreflected light 334 is collected into an input 335 of the imagingspectrometer 332.

[0203] At the microarray chip 328, redirected light 330 illuminates animaging area 332 along a bottom surface 342 of the microarray chip 328.The redirected light 330 is used to illuminate this imaging area 332. Inthis embodiment, the illuminated imaging area 332 may be defined by aplurality of spotw 331 along the microarray chip 328. Preferably, theilluminated imaging area 332 is defined by either a complete row or acomplete column of spotw contained on the microarray chip 328. Forexample, illuminated imaging area 332 could be defined to include thefirst microarray chip row 331 or row 333.

[0204] The bottom surface 342 of the microarray chip 328 is scanned in asequential manner. To accomplish a complete scan of the bottom surface342, the microarray chip 328 is transported along a scan direction “A.”As the microarray chip 328 is transported along this scan direction, thecollimated light 330 traverses along the complete length of the bottomsurface 342 of the microarray chip. In this manner, the instrumentsystem 319 sequentially illuminates and reads out all of the spotw in amicroarray chip 328. For example, as the chip 328 is moved in the “A”scan direction, chip row 333 is first illuminated and read out, and thenchip row 331 is illuminated and then read out. The process continuesuntil all rows are read.

[0205] Preferably, the illuminated imaging area includes all the spotwalong a microarray row. For example, at a given point in time, theredirected light may illuminate a first row of spotw on the 8×6microarray chip. Consequently, light 330 illuminates all six spotw331(a-f) contained in the first row 331 of the microarray chip 328. Asthe microarray chip 328 is transported along scan direction A, thecollimated white light source 320 simultaneously illuminates all of thespotw contained in the next microarray row. To complete the read out ofthe entire microarray chip, each of the eight (8) rows of the microarraychip are sequentially illuminated.

[0206] The spectrometer unit 332 preferably comprises an imagingspectrometer containing a two-dimensional Charge Coupled Device (CCD)camera and a diffraction grating. The reflected light 334 containing thebiosensor resonance signal for each spot is diffracted by the grating inthe spectrometer unit. The diffraction produces a spatially segregatedwavelength spectra for each point within the illuminated area. (See,e.g., FIG. 18(c). The wavelength spectrum has a second spatial componentcorresponding to the direction transverse to the scan direction “A.”This second spatial component is subdivided into discrete portionscorresponding to spotw in this transverse direction.

[0207] For example, if the imaging spectrometer includes a CCD camerathat contains 512×2048 imaging elements, then an illuminating line isspatially segregated into 512 imaging elements or points. A wavelengthspectra is measured for each of the 512 imaging elements or points alongthe orthogonal axis of the CCD camera. Where the CCD camera contains512×2048 imaging elements, the CCD would have a resolution of 2048wavelength data points. Using this method, the PWV's of 512 points aredetermined for a single “line” or imaging area across the sensor bottomsurface 342. For a conventional CCD imaging camera typically havingspatial resolution of approximately 10 microns, a 1:1 imaging system iscapable of resolving PWV values on sensor surface 342 with a 10 micronresolution. In order to measure a PWV image of the entire sensor bottomsurface 342, the sensor 328 is transported along an imaging plane (scandirection A), and subsequent line scans are used to construct a PWVimage.

[0208] FIGS. 21-24 illustrate various aspects of yet another alternativeembodiment of a measuring instrument 400 which incorporates variousprinciples of FIG. 19. FIG. 21 illustrates a perspective view of themeasuring instrument 400.

[0209] Measuring instrument 400 includes a measuring instrument cover452 and a door 454. A microplate well plate (or microtiter plate) 456configured as a biosensor in accordance with this invention is shown inan extracted position, outside an incubator assembly 460 incorporated inthe measuring instrument 400. The microplate well plate 456 is held by amicrowell tray 458. The tray 458 may extend out of the incubatorassembly 460 through a door way 453 located at the front of theincubator assembly 460. The incubator assembly 460 allows the tray 458to be maintained at a user defined temperature during microwell trayread out and/or measurement.

[0210] In one preferred embodiment, incubator assembly 460 is used forperforming assays at controlled temperatures, typically such controlledtemperatures may range from 4 and 45 degrees Celsius. As will beexplained with reference to FIGS. 22, 23, and 24, a collimator assembly708 is positioned preferably beneath a bottom portion 602 of theincubator assembly 460. During microtiter well illumination andwavelength measurement, the collimator assembly 708 illuminates a bottomsurface 459 of the tray 458.

[0211] While the tray 458 remains in an extracted position outside ofthe incubator assembly 460, the microtiter plate 456 may be placed on orremoved from the tray 458. The plate 456 may be held in the tray 458 viaa set of registration points, spring clips, or other known types ofsecuring means. In FIG. 22, clips 457 are used to hold the plate 456 inthe tray 458.

[0212] After the microtiter plate 456 has been loaded with a fluidsample with biological material to be detected and measured, the tray458 is transported into the incubator assembly 460. Processing, mixing,heating, and/or readout of the biosensors may then begin, preferablyunder the control of the controller assembly 588 (see FIG. 22).

[0213] Once the tray 458 retracts into the incubator assembly 460, thetray remains stationary during illumination and read out. For a readoutof the microtiter plate 456 to occur, the collimator assembly 708generates an illumination pattern that is incident along the bottomsurface 459 of the plate 456. Preferably, the measuring apparatus 400generates a beam of light that is incident along an entire row of wellsof the plate 456.

[0214] Alternatively, the measuring apparatus 400 generates a pluralityof illumination beams that are simultaneously incident on a plurality ofplate wells. The illumination pattern, comprising multiple beams, isgenerated by dual illumination fiber optic probes contained within thecollimator assembly 708. The construction of the probes is as shown isFIG. 17. As previously herein described, the light reflected off of thebiosensor surface may then be detected by the same plurality of probescontained within a collimator assembly 708. This reflected light is thenanalyzed via the spectrometer system 590.

[0215] The incubator assembly 460 is provided with a plurality ofapertures 764 along a bottom incubator assembly structure. As can beseen in FIG. 24, incubator assembly apertures 764 are configured togenerally line-up and match the well locations 657 on the plate 456 whenthe plate 456 is in a readout position within the incubator assembly460. For example, if there are 96 wells on the microwell well plate 456,the incubator assembly bottom portion 602 will be provided with 96apertures 764. These apertures will be configured in the same type ofarray as the wells of the well plate (e.g., 8 rows by 6 columns). Theseapertures 764 provide clearance for light generated by collimatorassembly 708 to reach the wells from the illuminating probes 709.

[0216] To enable user access to the tray and to the plate, the platetray 458 extends out of the measuring apparatus 400. The tray 458 can beretracted into the apparatus 400 and the door cover 454 closed to beginmicroplate processing. Such processing could include mixing liquid inthe microtiter wells, heating deposited liquids to a predeterminedtemperature, illumination of the microplate 456, and processing variousreflected illumination patterns.

[0217]FIG. 22 illustrates a perspective view 580 of various internalcomponents of the measuring instrument 400 illustrated in FIG. 21. Asshown in FIG. 22, internal components of the measuring instrument 580include a transition stage assembly 560, heater controller unit 582, acontroller board assembly 588, and a spectrometer unit 590. Thetransition stage assembly 560 includes the incubator assembly 460 andthe collimator assembly 708. The heater controller unit 582, thecontroller board assembly 588, the transition stage assembly 560, andthe spectrometer unit 590 are mounted on a base plate 592. Themicroplate well tray 556 is shown in the retracted position, outside ofthe incubator assembly 460.

[0218] The heater controller unit 582 provides temperature control tothe incubator assembly 460. The controller board assembly 588 providesfunctional controls for the measuring apparatus including the mixing andother motion controls related to translation stage 560 and tray handling458.

[0219] The spectrometer unit 590 contains an appropriate spectrometerfor generating the PWV data. The design of the spectrometer will varydepending on the illumination source. If the probes of FIG. 17 are used,the spectrometer will ordinarily have the design shown in FIG. 18B.

[0220]FIG. 23 illustrates a perspective view of the transition stageassembly 560 of the measuring instrument 400 illustrated in FIGS. 21 and22. FIG. 26 illustrates the transition stage assembly 560 of FIG. 23with an incubator assembly top portion 461 removed (See FIGS. 22 and23). As can be seen from FIGS. 23 and 24, the transition stage assembly560 includes the microwell tray 458 positioned in the retractedposition. The microwell tray 458 has a plurality of wells 657, entersthe incubation assembly 460 (FIG. 23) to initiate the read out process.

[0221] The microwell plate tray 458 is mounted on a top surface 605 of abottom portion 602 of the incubator assembly 460. Preferably, where themicrotiter tray 456 is a conventional microtiter tray having 96 wells,the bottom portion 602 of the incubator assembly 460 includes 96 holes.The microwell plate tray 458 is positioned over the bottom portion ofthe incubator assembly 602 such that the incubator assembly apparatusessentially matches up with the apertures (wells) contained in themicrowell tray 458. Alternately, bottom portion 602 may contain atransparent section that matches the bottom portion of the plate, or mayeliminate the bottom portion.

[0222] During specimen illumination and measurement, the microwell tray458 is preferably held in a stationary manner within the incubatorassembly 460 by the bottom incubator assembly portion 602. Duringillumination and measurement, the collimator assembly 708 is held in astationary manner while a stepping motor 606 drives the incubatorassembly, including the plate, in a linear direction “A”. As theincubator assembly 460 is driven along direction “A,” the collimatorassembly 708 illuminates the bottom surface 459 of microtiter plate 456.The resulting reflected illumination patterns are detected by thecollimator assembly 708. A home position sensor 710 is provided as aportion of the translation stage assembly and to determine the positionduring the illumination process.

[0223] The transition stage assembly 760 is provided with a plurality ofelastomer isolators 762. In this embodiment, a total of six elastomerisolators are used to provide isolation and noise reduction duringillumination and read out.

[0224] As can be seen from FIGS. 23 and 24, the collimator assembly 708is positioned below a bottom surface 603 of the incubator portion bottomportion 602. Preferably, the collimator assembly 708 includes aplurality of dual fiber probe heads 709. In the embodiment illustratedin FIG. 24, the collimator assembly 708 includes 8 dual fiber probeheads 709. These dual fiber probes could have a probe head configurationsimilar to the fiber optic probes illustrated in FIG. 19 and aspreviously described. Alternatively, the collimator assembly 708 couldinclude a PWV imaging system such as the PWV imaging system illustratedin FIG. 20.

[0225] For ease of explanation, only the bottom plate 602 of theincubator assembly 460 is shown is FIG. 24. The incubator assemblybottom portion 602 is provided with a plurality of apertures 764.Preferably, where the microwell plate 456 is provided with an 8×12 arrayof wells such as illustrated in FIG. 24, the incubator assembly bottomportion 602 will also include an 8×12 array of 96 apertures. Theseapertures will essentially match the 96 wells on the microwell plate456. In this manner, the collimated white light generated by thecollimator assembly 708 propagates through a first surface 603 along theincubator assembly bottom portion 602, and exit a second surface or topsurface 605 of incubator assembly bottom portion 602. The collimatedlight can then illuminate a bottom well portion of the microwell plate456. Alternately, bottom portion 602 may contain a transparent sectionthat matches the bottom portion of the plate, or may eliminate thebottom portion.

[0226] Referring to FIGS. 23 and 24, a drive motor 606 is provided fordriving the incubator assembly during well scanning. A home positionsensor 710 is provided as a stop measuring during the translation stage.The plate handling stage uses a stepping motor 702 to drive arack-and-pinion mechanism. The scanning stage uses a stepping motor 606to drive a leadscrew 559 along translation stage rails 557, 558.

[0227] A mixer assembly may be used for mixing the liquid in the wells.In the present invention, a mixing mechanism is located between theincubation chamber of the translation stage. Additionally, a mixingmechanism may be provided in an alternative location.

[0228]FIG. 25 illustrates an example of a microarray image.Specifically, FIG. 25 illustrates ten spots 800 of human-IgG spotted ona TaO sensor surface. Each spot is approximately 400-microns indiameter. FIG. 25 illustrates the result of subtracting a pre-spottedimage from a post-spotted image. The intensity scale conversion factoris illustrated to be a 0.04 nm per display intensity unit, resulting ina detected wavelength shift of 0.8 nm.

[0229] 11. Angular Scanning

[0230] The proposed detection systems are based on collimated whitelight illumination of a biosensor surface and optical spectroscopymeasurement of the resonance peak of the reflected beam. Molecularbinding on the surface of a biosensor is indicated by a shift in thepeak wavelength value, while an increase in the wavelength correspondsto an increase in molecular absorption.

[0231] As shown in theoretical modeling and experimental data, theresonance peak wavelength is strongly dependent on the incident angle ofthe detection light beam. Because of the angular dependence of theresonance peak wavelength, the incident white light needs to becollimated. Angular dispersion of the light beam broadens the resonancepeak, and could reduce biosensor detection sensitivity. In addition, thesignal quality from the spectroscopic measurement could depend on thepower of the light source and the sensitivity of the detector. In orderto obtain a desirable signal-to-noise ratio, a lengthy integration timefor each detection location may be required, and therefore lengthenoverall time to readout a biosensor plate. A tunable laser source can beused for detection of grating resonance, but is generally costprohibitive.

[0232] In one embodiment, these disadvantages are addressed by using alaser beam for illumination of a biosensor, and a light detector formeasurement of reflected beam power. A scanning mirror device can beused for varying the incident angle of the laser beam, and an opticalsystem is used for maintaining collimation of the incident laser beam.See, e.g., “Optical Scanning” (Gerald F. Marchall ed., Marcel Dekker(1991). Any type of laser scanning can be used. For example, a scanningdevice that can generate scan lines at a rate of about 2 lines to about1,000 lines per second is useful in the invention. In one embodiment ofthe invention, a scanning device scans from about 50 lines to about 300lines per second.

[0233] In one embodiment, the reflected light beam passes through partof the laser scanning optical system, and is measured by a single lightdetector. The laser source can be a diode laser with a wavelength of,for example, 780 nm, 785 nm, 810 nm, or 830 nm. Laser diodes such asthese are readily available at power levels up to 150 mW, and theirwavelengths correspond to high sensitivity of Si photodiodes. Thedetector thus can be based on photodiode biosensors.

[0234] In another detection system embodiment, while a scanning mirrorchanges its angular position, the incident angle of the laser beam onthe surface changes by nominally twice the mirror angular displacement.The scanning mirror device can be a linear galvanometer, operating at afrequency of about 2 Hz up to about 120 Hz, and mechanical scan angle ofabout 10 degrees to about 20 degrees. In this example, a single scan canbe completed within about 10 msec. A resonant galvanometer or a polygonscanner can also be used.

[0235] The angular resolution depends on the galvanometer specification,and reflected light sampling frequency. Assuming galvanometer resolutionof 30 arcsec mechanical, corresponding resolution for biosensor angularscan is 60 arcsec, i.e. 0.017 degree. In addition, assume a samplingrate of 100 ksamples/sec, and 20 degrees scan within 10 msec. As aresult, the quantization step is 20 degrees for 1000 samples, i.e. 0.02degree per sample. In this example, a resonance peak width of 0.2degree, as shown by Peng and Morris (Experimental demonstration ofresonant anomalies in diffraction from two-dimensional gratings, OpticsLett., 21:549 (1996)), will be covered by 10 data points, each of whichcorresponds to resolution of the detection system.

[0236] The advantages of such a detection system includes: increasedcollimation of incident light by a laser beam, high signal-to-noiseratio due to high beam power of a laser diode, low cost due to a singleelement light detector instead of a spectrometer, and high resolution ofresonance peak due to angular scanning.

[0237] 12. Mathematical Resonant Peak Determination

[0238] The sensitivity of a biosensor is determined by the shift in thelocation of the resonant peak when material is bound to the biosensorsurface. Because of noise inherent in the spectrum, it is preferable touse a procedure for determining an analytical curve—the turning point(i.e., peak) of which is well defined. Furthermore, the peakcorresponding to an analytic expression can be preferably determined togreater than sub-sampling-interval accuracy, providing even greatersensitivity.

[0239] One embodiment utilizes a method for determining a location of aresonant peak for a binding partner in a resonant reflectance spectrumwith a colormetric resonant biosensor. The method comprises selecting aset of resonant reflectance data for a plurality of colormetric resonantbiosensors or a plurality of biosensor distinct locations. The set ofresonant reflectance data is collected by illuminating a colormetricresonant diffractive grating surface with a light source and measuringreflected light at a pre-determined incidence. The colormetric resonantdiffractive grating surface is used as a surface binding platform forone or more specific binding substances such that binding partners canbe detected without use of a molecular label.

[0240] The step of selecting a set of resonant reflectance data caninclude selecting a set of resonant reflectance data:

[0241] a. x₁ and y₁ for i=1, 2, 3, . . . n,

[0242] b. wherein x₁ is a first measurement includes a first reflectancespectra of one or more specific binding substances attached to thecolormetric resonant diffractive grating surface, y₁ is a secondmeasurement and includes a second reflectance spectra of the one or morespecific binding substances after a plurality of binding partners areapplied to colormetric resonant diffractive grating surface includingthe one or more specific binding substances, and n is a total number ofmeasurements collected.

[0243] The set of resonant reflectance data includes a plurality of setsof two measurements, where a first measurement includes a firstreflectance spectra of one or more specific binding substances that areattached to the colormetric resonant diffractive grating surface and asecond measurement includes a second reflectance spectra of the one ormore specific binding substances after one or more binding partners areapplied to the colormetric resonant diffractive grating surfaceincluding the one or more specific binding substances. A difference in apeak wavelength between the first and second measurement is ameasurement of an amount of binding partners that bound to the one ormore specific binding substances. A sensitivity of a colormetricresonant biosensor can be determined by a shift in a location of aresonant peak in the plurality of sets of two measurements in the set ofresonant reflectance data.

[0244] A maximum value for a second measurement from the plurality ofsets of two measurements is determined from the set of resonantreflectance data for the plurality of binding partners, wherein themaximum value includes inherent noise included in the resonantreflectance data. A maximum value for a second measurement can includedetermining a maximum value y_(k) such that:

[0245] c. (y_(k)>=y₁) for all i,≠k.

[0246] The algorithm determines whether the maximum value is greaterthan a pre-determined threshold. This can be calculated by, for example,computing a mean of the set of resonant reflectance data; computing astandard deviation of the set of resonant reflectance data; anddetermining whether ((y_(k)−mean)/standard deviation) is greater than apre-determined threshold. The pre-determined threshold is determined bythe user. The user will determine what amount of sensitivity is desiredand will set the pre-determined threshold accordingly.

[0247] If the maximum value is greater than a pre-determined threshold acurve-fit region around the determined maximum value is defined. Thestep of defining a curve-fit region around the determined maximum valuecan include, for example:

[0248] d. defining a curve-fit region of (2w+1) bins, wherein w is apre-determined accuracy value;

[0249] e. extracting (x₁, k−w<=i<=k+w); and

[0250] f. extracting (y₁, k−w<=i<=k+w).

[0251] A curve-fitting procedure is performed to fit a curve around thecurve-fit region, wherein the curve-fitting procedure removes apre-determined amount of inherent noise included in the resonantreflectance data. A curve-fitting procedure can include, for example:

[0252] g. computing g₁=ln y₁;

[0253] h. performing a 2^(nd) order polynomial fit on g₁ to obtain g′₁defined on

[0254] i. (x₁,k−w<=i<=k+w);

[0255] j. determining from the 2^(nd) order polynomial fit coefficientsa, b and c of for (ax²+bx+c)-; and

[0256] k. computing y′_(i)=e^(g′1).

[0257] The location of a maximum resonant peak is determined on thefitted curve, which can include, for example, determining a location ofmaximum reasonant peak (x_(p)=(−b)/2a). A value of the maximum resonantpeak is determined, wherein the value of the maximum resonant peak isused to identify an amount of biomolecular binding of the one or morespecific binding substances to the one or more binding partners. A valueof the maximum resonant peak can include, for example, determining thevalue with of x_(p) at y′_(p)

[0258] Alternatively, peak values of the measurement apparatusembodiments may be derived by the mathematical resonant peakdetermination described in commonly assigned related copending patentapplication Ser. No. ______ (MBHB 01-1775), the entirety of which isherein incorporated by reference and to which the reader is directed forfurther information.

[0259] One embodiment of the measurement apparatus includes a computerreadable medium having stored therein instructions for causing aprocessor to execute a method for determining a location of a resonantpeak for a binding partner in a resonant reflectance spectrum with acolormetric resonant biosensor. A computer readable medium can include,for example, magnetic disks, optical disks, organic memory, and anyother volatile (e.g., Random Access Memory (“RAM”)) or non-volatile(e.g., Read-Only Memory (“ROM”)) mass storage system readable by theprocessor. The computer readable medium includes cooperating orinterconnected computer readable medium, which exist exclusively on aprocessing system or to be distributed among multiple interconnectedprocessing systems that can be local or remote to the processing system.

[0260] The following are provided for exemplification purpose only andare not intended to limit the scope of the invention described in broadterms above. All references cited in this disclosure are incorporatedherein by reference.

[0261] Sensor Readout Instrumentation

[0262] In order to detect reflected resonance, a white light source canilluminate an approximately 1 mm diameter region of a biosensor surfacethrough a 400 micrometer diameter fiber optic and a collimating lens.Smaller or larger areas may be sampled through the use of illuminationapertures and different lenses. A group of six detection fibers may bebundled around the illumination fiber for gathering reflected light foranalysis with a spectrometer (Ocean Optics, Dunedin, Fla.). For example,a spectrometer can be centered at a wavelength of 800 nm, with aresolution of approximately 0.14 nm between sampling bins. Thespectrometer integrates reflected signal for 25-75 msec for eachmeasurement. The biosensor sits upon an x-y motion stage so thatdifferent regions of the biosensor surface can be addressed in sequence.

[0263] Equivalent measurements can be made by either illuminating thetop surface of device, or by illuminating through the bottom surface ofthe transparent substrate. Illumination through the back is preferredwhen the biosensor surface is immersed in liquid, and is most compatiblewith measurement of the biosensor when it is incorporated into thebottom surface of, for example, a microwell plate.

[0264] Mathematical Resonant Peak Determination

[0265] This example discusses some of the findings that have beenobtained from looking at fitting different types of curves to theobserved data.

[0266] The first analytic curve examined is a second-order polynomial,given by

y=ax ² +bx+c

[0267] The least-squares solution to this equation is given by the costfunction${\varphi = {\sum\limits_{i = 1}^{n}\quad \left( {{ax}_{i}^{2} + {bx}_{i} + c - y_{i}} \right)^{2}}},$

[0268] the minimization of which is imposed by the constraints$\frac{\partial\varphi}{\partial a} = {\frac{\partial\varphi}{\partial b} = {\frac{\partial\varphi}{\partial b} = 0.}}$

[0269] Solving these constraints for a, b, and c yields $\begin{pmatrix}a \\b \\c\end{pmatrix} = {\begin{pmatrix}{\sum\quad x_{i}^{4}} & {\sum\quad x_{i}^{3}} & {\sum\quad x_{i}^{2}} \\{\sum\quad x_{i}^{3}} & {\sum\quad x_{i}^{2}} & {\sum\quad x_{i}} \\{\sum\quad {x2}} & {\sum\quad x_{i}} & n\end{pmatrix}^{- 1} \cdot {\begin{pmatrix}{\sum\quad {x_{i}^{2}y_{i}}} \\{\sum\quad {x_{i}y_{i}}} \\{\sum\quad y_{i}}\end{pmatrix}.}}$

[0270] Empirically, the fitted curve does not appear to have sufficientrise and fall near the peak. An analytic curve that provides bettercharacteristics in this regard is an exponential curve, such as aGaussian curve. A simple method for performing a Gaussian-like fit is toassume that the form of the curve is given by

y=e^(ax) ² ^(+bx+c),

[0271] in which case the quadratic equations above can be utilized byforming y′, where y′=lny.

[0272] Assuming that the exponential curve is the preferred data fittingmethod, the robustness of the curve fit is examined in two ways: withrespect to shifts in the wavelength and with respect to errors in thesignal amplitude.

[0273] To examine the sensitivity of the analytical peak location whenthe window from which the curve fitting is performed is altered to fall10 sampling intervals to the left or to the right of the true maxima.The resulting shift in mathematically-determined peak location is shownin Table 3. The conclusion to be derived is that the peak location isreasonably robust with respect to the particular window chosen: for ashift of approximately 1.5 nm, the corresponding peak location changedby only <0.06 nm, or 4 parts in one hundred sensitivity.

[0274] To examine the sensitivity of the peak location with respect tonoise in the data, a signal free of noise must be defined, and thenincremental amounts of noise is added to the signal and the impact ofthis noise on the peak location is examined. The ideal signal, forpurposes of this experiment, is the average of 10 resonant spectraacquisitions.

[0275] Gaussian noise of varying degrees is superimposed on the idealsignal. For each such manufactured noisy signal, the peak location isestimated using the 2^(nd)-order exponential curve fit. This is repeated25 times, so that the average, maximum, and minimum peak locations aretabulated. This is repeated for a wide range of noise variances—from avariance of 0 to a variance of 750. TABLE 3 Comparison of peak locationas a function of window location Shift Window Peak Location □ = −10 bins771.25-782.79 nm 778.8221 nm □ = 0 bins 772.70-784.23 nm 778.8887 nm □ =+10 bins 774.15-785.65 nm 7778.9653 nm

[0276] The conclusion of this experiment is that the peak locationestimation routine is extremely robust to noisy signals. In oneembodiment, the entire range of peak locations is only 1.5 nm, even withas much random noise variance of 750 superimposed—an amount of noisethat is substantially greater that what has been observed on thebiosensor thus far. The average peak location, despite the level ofnoise, is within 0.1 nm of the ideal location.

[0277] Based on these results, a basic algorithm for mathematicallydetermining the peak location of a colorimetric resonant biosensor is asfollows:

[0278] 1. Input data x_(l) and y_(l), i=1, . . . ,n

[0279] 2. Find maximum

[0280] a. Find k such that y_(k)≧y_(l) for all i≠k

[0281] 3. Check that maximum is sufficiently high

[0282] a. Compute mean {overscore (y)} and standard deviation σ ofsample

[0283] b. Continue only if (y_(k)−{overscore (y)})/σ>UserThreshold

[0284] 4. Define curve-fit region of 2w+1 bins (w defined by the user)

[0285] a. Extract x_(l),k−w≦i≦k+w

[0286] b. Extract y_(l),k−w≦i≦k+w

[0287] 5. Curve fit

[0288] a. g_(l)=ln y_(l)

[0289] b. Perform 2^(nd)-order polynomial fit to obtain g′_(i) definedon x_(l),k−w≦i≦k+w

[0290] c. Polynomial fit returns coefficients a,b,c of form ax²+bx+c

[0291] d. Exponentiate: y′_(l)=e^(g′) ^(_(l))

[0292] 6. Output

[0293] a. Peak location p given by x_(p)=−b/2a

[0294] b. Peak value given by y′_(p)(x_(p))

[0295] In summary, a robust peak determination routine has beendemonstrated; the statistical results indicate significant insensitivityto the noise in the signal, as well as to the windowing procedure thatis used. These results lead to the conclusion that, with reasonablenoise statistics, that the peak location can be consistently determinedin a majority of cases to within a fraction of a nm, perhaps as low as0.1 to 0.05 nm.

[0296] Those skilled in the art to which the present invention pertainsmay make modifications resulting in other embodiments employingprinciples of the present invention without departing from its spirit orcharacteristics, particularly upon considering the foregoing teachings.Accordingly, the described embodiments are to be considered in allrespects only as illustrative, and not restrictive, and the scope of thepresent invention is, therefore, indicated by the appended claims ratherthan by the foregoing description. Consequently, while the presentinvention has been described with reference to particular embodiments,modifications of structure, sequence, materials and the like apparent tothose skilled in the art would still fall within the scope of theinvention.

We claim:
 1. An instrument system for detecting a biochemicalinteraction on a biosensor comprising an array of detection locations,said system comprising: a light source for generating collimated whitelight; a beam splitter directing said collimated white light towards asurface of a sensor corresponding to said detector locations; and adetection system including an imaging spectrometer receiving saidreflected light and generating an image of said reflected light.
 2. Theinvention of claim 1 wherein said biosensor is a microarray chip.
 3. Theinvention of claim 2 wherein said microarray chip is a conventionalmicroarray chip.
 4. The invention of claim 1 wherein said biosensor istransported along a scan direction.
 5. The invention of claim 1 whereinsaid imaging spectrometer generates a Peak Wavelength Value image. 6.The invention of claim 1 wherein said directed collimated white light isdirected to a plurality of locations on said surface of said sensor. 7.The invention of claim 1 wherein said directed collimated white light issimultaneously directed to a plurality of locations on said surface ofsaid sensor.
 8. The invention of claim 1 wherein said directedcollimated white light is directed to an imaging area on said surface ofsaid sensor.
 9. The invention of claim 2 wherein said directedcollimated white light illuminates a plurality of wells of saidmicroarray chip.
 10. The invention of claim 1 wherein said imagingspectrometer includes a two-dimensional Charge Coupled Device camera.11. The invention of claim 1 wherein said imaging spectrometer includesa diffraction grating.
 12. The invention of claim 1 including a softwareinterface, said software interface controlling said imagingspectrometer.
 13. The invention of claim 12 wherein said softwareinterface coordinates Peak Wavelength Value determination with an x-ymotion stage.
 14. The invention of claim 12 wherein said softwareinterface converts measured data into a Peak Wavelength Value.
 15. Theinvention of claim 1, wherein said light source illuminates saidbiosensor from a sensor top surface
 16. The invention of claim 1 whereinsaid light source illuminates said biosensor from a sensor bottomsurface.
 17. An instrument for calculating a peak wavelength, saidinstrument comprising: an incubator assembly for incubating a biosensor;an optical assembly, said optical assembly illuminating said biosensorwith light and collecting reflected radiation from said biosensor; aspectrometer receiving said reflected radiation; and software deriving apeak wavelength from said reflected and detected wavelength.
 18. Theinvention of claim 17 wherein said biosensor is embedded within a bottomportion of a microtiter plate.
 19. The invention of claim 17 whereinsaid collimator assembly comprises a plurality of fiber optic probes.20. The invention of claim 18 wherein said plurality of fiber opticprobes comprise an illuminating fiber optic probe for illuminating saidbiosensor and a detecting fiber optic probe for detecting said reflectedwavelength.
 21. The invention of claim 18 wherein said collimatorassembly comprises a beam splitter, said beam splitter enables saidilluminated light and said reflected light to share a common opticalpath.
 22. The invention of claim 17 wherein said collimator assemblyincludes a collimating lens, said collimating lens focuses said whitelight on said biosensor surface.
 23. The invention of claim 17 whereinsaid biosensor comprises: a first two-dimensional grating comprising afirst refractive index material and having a top surface and a bottomsurface; a substrate layer comprising a top surface and a bottomsurface, wherein said top surface of said substrate supports said bottomsurface of said first two-dimensional grating; and a secondtwo-dimensional grating comprising a second refractive index materialand having a top surface and a bottom surface, wherein said bottomsurface of said second two-dimensional grating is attached to saidbottom surface of said substrate; wherein, when said biosensor isilluminated two resonant grating effects are produced in said reflectedradiation spectrum, and wherein said depth and period of both of saidtwo-dimensional gratings are less than said wavelength of said resonantgrating effects.